Note: Descriptions are shown in the official language in which they were submitted.
~
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IMPROVED METHOD FOR THE BIOSYNTHESIS OF VITAMIN E
The invention relates to improved processes for the biosynthesis
of vitamine E. These processes are characterized by inhibiting
homogentisate (HG) breakdown via maleyl acetoacetate (MAA),
fumaryl acetoacetate (FAA) to give fumarate and acetoacetate.
Also in accordance with the invention is the combination of this
inhibition with processes which further increase the supply of
homogentisate, or which promote the conversion of homogentisate
into vitamin E.
35
Homogentisate is an important metabolite. It is a degradation
product of the amino acids tyrosine and phenylalanine. In humans'
15 and animals, homogentisate is broken down further to maleyl
acetoacetate, subsequently to fumaryl acetoacetate and then, into
fumarate and acetoacetate. Plants and other photosynthesizing
microorganism furthermore utilize homogentisate as starting
material for the synthesis of tocopherols and tocotrienols.
The naturally occurring eight compounds with vitamin E activity
are derivatives of 6-chromanol (Ullmann's Encyclopedia of
Industrial Chemistry, Vol. A 27 (1996), VCH Verlagsgesellschaft,
Chapter 4., 478-488, vitamin E). The tocopherol group (1a-d) has
a saturated side chain, while the tocotrienol group (2a-d) has an
unsaturated side chain:
R1
HO
2 ~ / ~1)
1a, Ct-tocopherol: R1 = R2 = R3 = CH3
1b, (3-tocopherol [ 148-03-8 ] : Rl = R3 = CH3 , R2 = H
lc, 'y-tocopherol [54-28-4] : Rl = H, R2 = R3 = CH3
1d, 8-tocopherol (119-13-1]: R1 = RZ = H, R3 = CH3
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2
1
H
2a, cc-tocotrienol[1721-51-3] = R2 = R3 = CH3
: R1
(3-tocotrienol[490-23-3] : R3 = CH3, R2
2b, Rl = = H
2c, y-tocotrienol[14101-61-2] = H, R2 = R3
: Rl = CH3
2d, 8-tocotrienol[25612-59-3]: = R2 = H, R3
R1 = CH3
(2)
For the purposes of the present invention, vitamin E is to be
understood as meaning all of the eight abovementioned tocopherols
and tocotrienols with vitamin E activity.
These compounds with vitamin E activity are important natural
lipid-soluble antioxidants. Vitamin E deficiency leads to
pathophysiological situations in humans and animals. It has been
revealed in epidemiological studies that food supplementation
with vitamin E reduces the risk of developing cardiovascular
diseases or cancer. Furthermore, a positive effect on the immune
system and the prevention of general age-related degenerative
symptoms have been described (Traber MG, Sies H; Annu Rev Nutr.
1996;16:321-47). The function of vitamin E is probably a
stabilization of the biomembranes and a reduction of free
radicals as they are formed, for example, upon the lipid
oxidation of polyunsaturated fatty acids (PUFAs).
Little work has gone into studying the function of vitamin E in
the plants themselves. Possibly, however, it seems to play an
important role in the stress response of the plant, in particular
oxidative stress. Increased vitamin E levels were linked to
improved stability and shelf life of plant-derived products. The
supplementation with vitamin E of animal nutrition products has a
positive effect on meat quality and the shelf life of the meat
and meat products in, for example, pigs, cattle and poultry.
Thus, vitamin E compounds are of great economic value as
additives in the food and feed sectors, in pharmaceutical
formulations and in cosmetic applications.
In nature, vitamin E is synthesized exclusively by plants and
other photosynthetically active organisms (for example
cyanobacteria). The vitamin E content varies greatly. Most of the
staple food plants (for example wheat, rice, maize, potato) only
0817/00017
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have a very low vitamin E content (Hess, Vitamin E, a-tocopherol,
In Antioxidants in Higher Plants, editors: R.Ascher and J.Hess,
1993, CRC Press, Boca Raton, pp. 111-134). As a rule, oil crops
have a markedly higher vitamin E content, with a-, ~- and
8-tocopherol dominating. The recommended daily dose of vitamin E
is 15-30 mg.
Fig. 1 shows a biosynthetic scheme of tocopherols and
tocotrienols.
During biosynthesis, homogentisic acid (hornogentisate; HG) is
bound to phytyl pyrophosphate (PPP) or geranylgeranyl
pyrophosphate in order to form the precursors of a-tocopherol and
a-tocotrienol, namely 2-methylphytylhydroquinone and
2-methylgeranylgeranyl hydroquinone, respectively. Methylation
steps with S-adenosylmethionine as methyl donor first gives
2,3-dimethyl-6-phytylhydroquinone, cyclization then gives
~-tocopherol, and further methylation gives a-tocopherol.
Furthermore, ~- and 8-tocopherol can be synthesized by methylation
of 2-methylphytylhydroquinone.
Little is known as yet about increasing the metabolite flux to
increase the tocopherol or tocotrienol content in transgenic
organisms, for example in transgenic plants, by overexpressing
individual biosynthesis genes.
WO 97/27285 describes a modification of the tocopherol content by
increased expression or by downregulation of the enzyme
p-hydroxyphenyl-pyruvate dioxygenase (HPPD).
WO 99/04622 describes gene sequences encoding a y-t.ocopherol
methyltransferase from Synechocystis PCC6803 and Arabidopsis
thaliana, and its incorporation into transgenic plants.
WO 99/23231 demonstrates that the expression of a geranylgeranyl
oxidoreductase in transgenic plants results in an increased
tocopherol biosynthesis.
WO 00/10380 shows a modification of the vitamin E composition
using 2-methyl-6-phytylplastoquinol methyltransferase.
It has been shown by Shintani and DellaPenna that overexpression
of ~-tocopherol methyltransferase can markedly increase the
vitamin E content (Shintani and Dellapenna, Science 282
(5396):2098-2100, 1998).
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All reactions of vitamin E biosynthesis involve homogentisate. As
yet, most studies have concentrated on the overexpression of
genes of vitamin E or homogentisate biosynthesis (see above). The
competing reactions which break down homogentisage and thus
remove it from vitamin E biosynthesis have received little
attention to date.
The breakdown of homogentisate via maleyl acetoacetate and
fumaryl acetoacetate into fumarate and acetoacetate has been
described for nonphotosynthetically active organisms, mainly
animal organisms (Fernandez-Canon JM et al., Proc Natl Acad Sci
USA. 1995; 92 (20):9132-9136j. Animal organisms exploit this
metabolic pathway .for breaking down aromatic amino acids which
are predominantly ingested with the food. Its function and
relevance in plants, in contrast, is unclear. The reactions are
catalyzed by homogentisate 1,2-dioxygenase (HGD; EC No.:
1.13.11.5), maleyl-acetoacetate isomerase (MAAI; EC No.:
5.2.1.2.) and fumaryl acetoacetate hydrolase (FAAH; EC No.:
3.7.1.2).
The Arabidopsis thaliana homogentisate 1,2-dioxygenase (HGD) gene
is known (Genbank Acc.-No. AF130845). Owing to a homology with
the Emericella nidulans fumaryl acetoacetate hydrolase
(gb~L41670), the Arabidopsis thaliana fumaryl acetoacetate
hydrolase gene had already been annotated as having similarity to
the former (Genbank Acc.-No. AC002131). However, express mention
may be made in the relevant Genbank entry that the annotation
alone is based on similarity and not on experimental data. The
Arabidopsis maleyl-acetoacetate isomerase (MAAI) gene was present
in Genbank as a gene (AC005312), but annotated as a putative
glutathione S-transferase. An Emericella nidulans MAAI was known
(Genbank Acc.-No. EN 1837).
In an abstract (Abstract No. 413) presented at the 1999 Annual
Meeting of the American Society of Plant Physiologists
(07.24.-28.1999, Baltimore, USA), Tsegaye et al. conjecture an
advantage in the combination of a cross of HPPD-overexpressing
plants with plants in which HGD is downregulated by an antisense
approach.
Despite some success, there continues to exist a demand for
optimizing vitamin E biosynthesis.
It is an object of the present invention to provide further
processes which influence the vitamin E biosynthetic pathway and
thus lead to further advantageous transgenic plants with an
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elevated vitamin E content.
We have found that this object is achieved by identifying the
homogentisate/maleyl acetoacetate/fumaryl acetoacetate/fumarate
5 catabolic pathway as essential competitive pathway for the
vitamin E biosynthetic pathway. We have found that inhibition of
this catabolic pathway results in an optimization of vitamin E
biosynthesis.
Accordingly, the present invention firstly relates to processes
for a vitamin E production by reducing the HGD, MAAI and/or FAAH
activity. A combination of the above-described inhibition of the
homogentisate catabolic pathway with other processes which lead
to an improved vitamin E biosynthesis by promoting the conversion
of homogentisate into vitamin E proves to be especially
advantageous. This can be realized by an increased supply of
reactants or by an increased reaction of homogentisate with
precisely these reactants. This effect can be achieved for
example by overexpressing homogentisate phytyltransferase (HGPT),
geranylgeranyl oxidoreductase, 2-methyl-6-phytylplastoquinol
methyltransferase or y-tocopherol methyltransferase.
A combination with genes which promote formation of
homogentisate, such as, for example, HPPD or the TyrA gene, is
furthermore advantageous.
Inhibition of the catabolic pathway from homogentisate via maleyl
acetoacetate and fumaryl acetoacetate to give fumarate and
acetatoacetate can be realized in a plurality of ways.
The invention relates to nucleic acid constructs comprising at
least one nucleic acid sequence (anti-MAAI/FAAH), which is
capable of inhibiting the maleyl acetoacetate/fumaryl
acetoacetate/fumarate pathway, or one of its functional
equivalents.
The invention furthermore relates to above-described nucleic acid
constructs which, besides the anti-MAAI/FAAH nucleic acid
sequence, additionally comprise at least one nucleic acid
sequence (pro-HG) which is capable of increasing the biosynthesis
of homogentisate (HG), or one of its functional equivalents, or
at least one nucleic acid sequence (pro-vitamin E} which is
capable of increasing vitamin E biosynthesis starting from
homogentisate, or one of its functional equivalents, or a
combination of pro-HG and pro-vitamin E, or their functional
equivalents.
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The invention furthermore relates to nucleic acid constructs
comprising a nucleic acid sequence (anti-HGD) which is capable of
inhibiting homogentisate 1,2-dioxygenase (HGD), or one of its
functional equivalents.
The invention furthermore relates to said anti-HGD nucleic acid
constructs which, besides the anti-HGD nucleic acid sequence,
additionally comprise at least one nucleic acid sequence encoding
a bifunctional chorismate mutase/prephenate dehydrogenase (TyrA),
or one of its functional equivalents, or at least one nucleic
acid sequence (pro-vitamin E), which is capable of increasing
vitamin E biosynthesis starting from homogentisate, or one of its
function equivalents, or a combination of pro-vitamin E and TyrA
sequences, or one of their functional equivalents.
TyrA encodes a bifunctional chorismate mutase/prephenate
dehydrogenase from E.coli, a hydroxyphenylpyruvate synthase
containing the enzymatic activities of a chorismate mutase and a
prephenate dehydrogenase which converts chorismate into
hydroxyphenyl pyruvate, the starting material for homogentisate
(Christendat D, Turnbull JL. Biochemistry. 1999 Apr
13;38(15):4782-93; Christopherson RI, Heyde E, Morrison JF.
Biochemistry. 1983 Mar 29;22(7):1650-6.).
The invention furthermore relates to nucleic acid constructs
comprising at least one nucleic acid sequence (pro-HG) which is
capable of increasing homogentisate (HG) biosynthesis, or one of
its functional equivalents, and at least one nucleic acid
sequence (pro-vitamin E) which is capable of increasing vitamin E
biosynthesis starting from homogentisate, or one of its
functional equivalents.
Also in accordance with the invention are functional analogs of
the abovementioned nucleic acid constructs. Functional analogs
means, in this context, for example a combination of the
individual nucleic acid sequences
1. on a polynucleotide (multiple constructs)
2. on several polynucleotides in one cell (cotransformation)
3. by crossing various transgenic plants, each of which
comprises at least one of said nucleotide sequences.
The nucleic acid sequences present in the nucleic acid construct
are preferably linked functionally to genetic control sequences.
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The transformation according to the invention of plants with a
pro-HG-encoding construct leads to an increased homogentise
formation. An undesirable efflux of this metabolite is avoided by
simultaneously transforming with anti-HGD, or anti-MAAI/FAAH, in
particular the anti-MAAI construct. Thus, an increased amount of
homogentisate is available in the transgenic plant for the
formation of vitamin E, for example, tocopherols, via the
intermediates methyl-6-phytylquinol and 2,3-dimethylphytylquinol
(cf. Fig. 1). Not only pro-HG, but also anti-MA.AI/FAAH or
anti-HGD, leads to an increased supply of homogentisate for
vitamin E biosynthesis. The conversion of homogentisate into
vitamin E can be improved by combined transformation with a
pro-vitamin-E-encoding construct and further increases the
biosynthes of vitamin E.
An "increase" in homogentisate biosynthesis is to be interpreted
broadly in this context and encompasses an increased
homogentisate (HG) biosynthese activity in the plant or the plant
part or tissue transformed with a pro-HG construct according to
the invention. A variety of strategies for increasing HG
biosynthesis activity are encompassed by the invention. The
skilled worker recognizes that a series of different methods is
available for influencing HG biosynthesis activity in the desired
fashion. The processes described subsequently are to be
understood as examples and not by way of limitation.
In the strategy which is preferred in accordance with the
invention, a nucleic acid sequence (pro-HG) is used which can be
transcribed and translated into a polypeptide which increases HG
biosynthesis activity. Examples of such nucleic acid sequences
are p-hydroxyphenyl-pyruvate dioxygenase (HPPD) from various
organisms, or the bacterial TyrA gene product. In addition to the
above-described artificial expression of known genes, it is also
possible to increase their activity by mutagenizing the
polypeptide sequence. Furthermore, increased transcription and
translation of the endogenous genes can be achieved, for example,
by using artificial transcription factors of the zinc finger
protein type (Beerli RR et al., Proc Natl Acad Sci U S A. 2000;
97 (4):1495-500). These factors attach to the regulatory regions
of the endogenous genes and cause expression or repression of the
endogenous gene, depending on how the factor is designed.
Especially preferred for pro-HG is the use of nucleic acids which
encode polypeptide of SEQ ID N0: 8, 11 or 16, especially
preferably nucleic acids with the sequences described by SEQ ID
NO: 7, 10 or 15.
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The "increase" in vitamin E biosynthesis activity is to be
understood in a similar fashion, genes being employed here whose
activity promote the conversion of homogentisate into vitamin E
(tocopherols, tocotrienols) or whose activity promotes the
synthesis of reactants of homogentisate such as, fox example,
phytyl pyrophosphate or geranylgeranyl pyrophosphate. Examples
which may be mentioned are homogentisate-phytyltransferase
(HGPT), geranylgeranyl oxidoreduktase,
2-methyl-6-phytylplastoquinol methyltransferase and Y-tocopherol
methyltransferase. Especially preferred is the use of nucleic
acids which encode polypeptides of SEQ ID N0: 14, 20, 22 or 24,
especially preferred are those with the sequences described by
SEQ ID NO: 13, 19, 21 or 23.
"Inhibition" is to be interpreted broadly in connection with
anti-MAAI/FAAH and/or anti-HGD and encompasses the partial, or
essentially complete, repression or blocking of the MAAI/FAAH
and/or HGD enzyme activity in the plant or the plant part or
tissue transformed with an anti-MAAI/FAAH and/or anti-HGD
construct according to the invention, which repression or
blocking is based on a variety of mechanisms in terms of cell
biology. Inhibition for the purposes of the invention also
encompasses a quantitative reduction of active HGD, MAAI or FAAH
in the plant up to an essentially complete absence of HGD, MAAI
or FAAH protein (i.e. absent detectability of HGD and/or MAAI or
F.AAH enzyme activity or absent immunological detectability of
HGD, MAAI or FAAH).
A variety of strategies for reducing or inhibiting the HGD or
MAAI or FAAH activity are encompassed by the invention. The
skilled worker recognizes that a series of different methods is
available for influencing the HGD or MAAI or FAAH gene expression
or enzyme activity in the desired manner.
The strategy which is preferred in accordance with the invention
encompasses the use of a nucleic acid sequence (anti-MAAI/FAAH
and/or anti-HGD) which can be transcribed into an antisense
nucleic acid sequence which is capable of inhibiting the HGD or
MAAI/FAAH activity, for example by inhibiting the expression of
endogenous HGD and/or MAAI or FAAH.
The anti-HGD and/or anti-MAAI/FAAH nucleic acid sequences
according to the invention can, in a preferred embodiment,
contain the coding nucleic acid sequence of HGD (anti-HGD) and/or
MAAI or FAAH (anti-MAAI/FAAH) inserted in antisense orientation,
or functional equivalent fragments of the sequences in question.
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Especially preferred anti-HGD nucleic acid sequences encompass
nucleic acid sequences which encode polypeptides comprising an
amino acid sequence of SEQ ID N0: 3 or functional equivalents
thereof. Especially preferred are nucleic acid sequences of SEQ
ID N0: 1, 2 or 12 or functional equivalents thereof.
Especially preferred anti-MAAI/FAAH nucleic acid sequences
encompass nucleic acid sequences which encode polypeptides
comprising an amino acid sequence of SEQ ID N0: 5 and 18 or
functional equivalents thereof. Especially preferred are nucleic
acid sequences of SEQ ID NO: 4, 6, 9 or 17 or functional
equivalents thereof, very especially preferred are the
part-sequences shown in SEQ ID NO: 41 or 42, or their functional
equivalents.
A preferred embodiment of the nucleic acid sequences according to
the invention encompasses an HGD, MAAI or FAAH sequence motif of
SEQ ID NO: 1, 2, 4, 6, 9, 12, 17, 41 or 42 in antisense
orientation. This leads to an increased transcription of nucleic
acid sequences in the transgenic plant which are complementary to
the endogenous coding HGD, MAAI or FAAH sequence or a part
thereof and which hybridize with this sequence at the DNA or RNA
level.
The antisense strategy can advantageously be combined with a
ribozyme method. Ribozymes are catalytically active RNA sequences
which, coupled to the antisense sequences, catalytically cleave
the target sequences (Tanner NK. FEMS Microbiol Rev. 1999;
23 (3):257-75). This can increase the efficacy of an anti-sense
strategy.
Further methods for inhibiting HGD and/or MAAI/FAAH expression
encompass the overexpression of homologous HGD and/or MAAI/FAAH
nucleic acid sequences, which leads to cosuppression (Jorgensen
et al., Plant Mol. Biol. 1996, 31 (5):957-973), induction of the
specific RNA breakdown by the plant with the aid of a viral
expression system (amplicon) (Angell, SM et al., Plant J. 1999,
20(3):357-362). These methods are also termed
"post-transcriptional gene silencing" (PTGS).
Further methods are the introduction of nonsense mutations into
the endogene by means of introducing RNA/DNA oligonucleotides
into the plant (Zhu et al., Nat. Biotechnol. 2000, 18(5):555-558)
or the generation of knockout mutants with the aid of, for
example, T-DNA mutagenesis (Koncz et al., Plant Mol. Biol. 1992,
20(5):963-976) or homologous recombination (Hohn, B. and
Puchta, H, Proc. Natl. Acad. Sci. USA. 1999, 96:8321-8323.).
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Furthermore, overexpression or repression of genes is also
possible using specific DNA-binding factors, for example the
abovementioned factors of the zinc finger transcription factor
type. Furthermore, factors may be introduced into a cell which
5 inhibit the target protein itself. The protein-binding factors
can be, for example, aptamers (Famulok M, and Mayer G. Curr Top
Microbiol Immunol. 1999; 243:123-36).
The above-described publications and the methods disclosed
10 therein for regulating plant gene expression are herewith
expressly referred to.
An anti-HGD and/or anti-MAAI/FAAH sequence for the purposes of
the present invention is thus selected in particular from among:
a) antisense nucleic acid sequences;
b) antisense nucleic acid sequences combined with a ribozyme
method
c) nucleic acid sequences encoding homologous HGD and/or
MAAI/FAAH and leading to cosuppresion;
d) viral nucleic acid sequences and expression constructs
causing HGD and/or MAAI/FAAH-RNA breakdown;
e) nonsense mutants of endogenous HGD- or MAAI/FAAH-encoding
nucleic acid sequences;
f) nucleic acid sequences encoding knockout mutants;
g) nucleic acid sequences which are suitable for homologous
recombination;
h) nucleic acid sequences encoding specific DNA- or
protein-binding factors with anti-HGD and/or anti-MAAI/FAAH
activity;
it being possible for the expression of each individual of these
anti-HGD or anti-MAAI/FAAH sequences to cause "inhibition" of the
HGD and/or MAAI/FAAH activity as defined for the invention. A
combined use of such sequences is also feasible.
A nucleic acid construct or nucleic acid sequence is to be
understood as meaning in accordance with the invention for
example a genomic or a complementary DNA sequence ox an RNA
sequence and semisynthetic or fully synthetic analogs thereof.
0817/00017
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These sequences can exist in linear or circular form,
extrachromosomally or integrated into the genome. The pro-HG,
pro-vitamin E, anti-HGD or anti-MAAI/FAAH nucleotide sequences of
the constructs according to the invention can be generated
synthetically or obtained naturally or comprise a mixture of
synthetic or natural DNA constituents and can be composed of
various heterologous HGD, MAAI/FAAH, pro-HG or pro-vitamin E gene
segments of various organisms. The anti-HGD and/or anti-MAAI/FAAH
sequence can be derived from one or more exons or introns, in
particular exons of the HGD, MAAI or FAAH genes.
Also suitable are artificial nucleic acid sequences as long as
they mediate the desired property, for example the increase in
the vitamin E content in the plant, by overexpression of at least
one pro-HG and/or pro-vitamin E gene and/or expression of an
anti-HDG and/or MAAI/FAAH sequence in crop plants, as described
above. For example, synthetic nucleotide sequences can be
generated which have codons which are preferred by the plants to
be transformed. These codons which are preferred by plants can be
determined in the customary manner from codons with the highest
protein frequency by referring to the codon usage. Such
artificial nucleotide sequences can be determined, for example,
by backtranslating proteins with HGD and/or MAAI/FAAH and/or
pro-HG activity or pro-vitamin E activity which have been
constructed by means of molecular modeling, or else by in-vitro
selection. Especially suitable are coding nucleotide sequences
which have been obtained by backtranslating a polypeptide
sequence in accordance with the codon usage which is specific for
the host plant. For example, to avoid undesired regulatory
mechanisms of the plant, DNA fragments can be backtranslated
starting from the amino acid sequence of a bacterial pro-HG, for
example the bacterial TyrA gene, taking into consideration the
codon usage of the plant, and the complete exogenous pro-HG
sequence can be generated therefrom for use in the plant. This is
used to express a pro-HG enzyme which is not, or only
insufficiently, subject to regulation by the plant, thus allowing
full overexpression of the enzyme activity.
All the abovementioned nucleotide sequences can be prepared in a
manner known per se by chemical synthesis starting from the
nucleotide units, for example by fragment condensation of
individual overlapping complementary nucletic acid units of the
double helix. Oligonucleotides can be synthesized chemically for
example in a known manner by the phosphoamidite method (Voet,
Voet, 2nd Edition, Wiley Press New York, page 896-897). When
preparing a nucleic acid construct, various DNA fragments can be
manipulated in such a way that a nucleotide sequence is obtained
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12
which reads in the correct direction and which has a correct
reading frame. To connect the nucleic acid fragments to each
other, adaptors or linkers can be added to the fragments. The
addition of synthetic oligonucleotides and filling in gaps with
the aid of the Klenow fragment of DNA polymerase and ligation
reactions and general cloning methods are described in Sambrook
et al. (1989), Molecular cloning: A laboratory manual, Cold
Spring Harbor Laboratory Press.
Functional equivalents of the pro-HG or pro-vitamin E sequences
are those sequences which, despite a deviating nucleotide
sequence, still encode a protein with the functions desired in
accordance with the invention, i.e. an enzyme whose activity
directly or indirectly increases the formation of homogentisate
(pro-HG), or an enzyme whose activity directly or indirectly
promotes the conversion of homogentisate to vitamin E
(pro-vitamin E).
Functional equivalents of anti-HGD and/or anti-MAAT/FAAH
encompass those nucleotide sequences which sufficiently repress
the HGD and/or MAAI/FAAH enzyme functions in the transgenic
plant. This can be effected for example by preventing or
repressing HGD and/or MAAI/FAAH processing, the transport of HGD
and/or MAAI/FAAH or their mRNA, inhibiting ribosome attachment,
inhibiting RNA splicing, inducing an RNA-degrading enzyme and/or
inhibiting translational elongation or termination. Direct
repression of the endogenous genes by DNA-binding factors, for
example of the zinc finger transcription factor type, is
furthermore possible. Direct inhibition of the polypeptides in
question, for example by aptamers, is also possible. Various
examples are given hereinabove.
Functional equivalents are also to be understood as meaning, in
particular, natural or artificial mutations of an originally
isolated sequence encoding HGD and/or MAAI/FAAH or pro-HG or
pro-vitamin E which continue to show the desired function.
Mutations encompass substitutions, additions, deletions,
exchanges or insertions of one or more nucleotide residues. Thus,
the present invention also encompasses, for example, those
nucleotide sequences which are obtained by modifying the HGD
and/or MAAI/FAAH and/or pro-HG or pro-vitamin E nucleotide
sequence. The purpose of such a modification may be, for example,
the further limitation of the coding sequence contained therein
or else, for example, the insertion of further restriction enzyme
cleavage sites or the removal of superfluous DNA.
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20
Techniques known per se, such as in-vitro mutagenesis, primer
repair, restriction or ligation may be used in cases where
insertions, deletions or substitutions such as, for example,
transitions and transversions, are suitable. Complementary ends
5 of the fragments may be provided for ligation by manipulations
such as, for example, restrictions, chewing-back or filling in
overhangs for blunt ends,
Substitution is to be understood as meaning the exchange of one
10 or more amino acids for one or more amino acids. Exchanges which
are preferably carried out are so-called conservative exchanges
where the replaced amino acid has a similar property as the
original amino acid, for example the exchange of Glu for Asp, Gln
for Asn, Val for Ile, Leu for Iie and Ser for Thr.
Deletion is the replacement of an amino acid by a direct bond.
Preferred positions for deletions are the termini of the
polypeptide and the linkages between the individual protein
domains.
Insertions are introductions of amino acids into the polypeptide
chain, a direct bond being formally replaced by one or more amino
acids.
25 Homology between two proteins is understood as meaning the
identity of the amino acids over in each case the entire length
of the protein which is calculated by comparison with the aid of
the program algorithm GAP (UWGCG, University of Wisconsin,
Genetic Computer Group) setting the following parameters:
Gap Weight: 12 Length Weight: 4
Average Match: 2.912 Average Mismatch: -2.003
Accordingly, a sequence which has at least 20~ homology of the
nucleic acid level with the sequence SEQ ID N0. 6 is to be
understood as meaning a sequence which, upon comparison of its
sequence with the sequence SEQ ID N0. 6 using the above program
algorithm with the above parameter set, has at least 20$
homology.
Functional equivalents derived from one of the nucleic acid
sequences used in the nucleic acid constructs or vectors
according to the invention, for example by substitution,
insertion or deletion of amino acids or nucleotides, have at
least 20~ homology, preferably 40~ homology, by preference at
0 817 / ~ ~ 017 CA 02422760 2003-03-17
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45
least 60~ homology, preferably at least 80$ homology, especially
preferably at least 90~ homology.
Further examples for the nucleic acid sequences employed in the
5 nucleic acid constructs or vectors according to the invention can
be found readily from various organisms whose genomic sequence is
known, such as, for example, Arabidopsis thaliana, by homology
alignments of the amino acid sequences or from the corresponding
backtranslated nucleic acid sequences from databases.
Functional equivalents also encompass those variants whose
function is reduced or increased compared to the starting gene or
gene fragment, i.e., for example, those pro-HG or pro-vitamin E
genes which encode a polypeptide variant with a lower or higher
enzymatic activity than that of the original gene.
Further suitable functionally equivalent nucleic acid sequences
which may be mentioned are sequences which encode fusion
proteins, part of the fusion protein being, for example, a pro-HG
or pro-vitamin E polypeptide or a functionally equivalent portion
thereof. The second portion of the fusion protein can be, for
example, a further polypeptide with enzymatic activity (for
example a further pro-HG or pro-vitamin E polypeptide or a
functionally equivalent portion thereof) or an antigenic
polypeptide sequence with the aid of which pro-HG or
pro-vitamin E expression can be detected (for example Myc tag or
His tag). However, they are preferably a regulatory protein
sequence such as, for example, a signal or transit peptide which
leads the pro-HG or pro-vitamin E protein to the desired site of
action.
The invention furthermore relates to recombinant vectors
comprising at least one nucleic acid construct in accordance with
the above definition, a nucleic acid sequence encoding an HGD,
MAAI or FAAH, or combinations of these options.
The nucleic acid sequences or nucleic acid constructs present in
the vectors are preferably linked functionally to genetic control
sequences.
Examples of vectors according to the invention may encompass
expression constructs of the following type:
a) 5'-plant-specific promoter/anti-HGD/terminator-3'
b) 5'-plant-specific promoter/anti-MAAI/FAAH/terminator-3'
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0817/00017
c) 5'-plant-specific promoter/pro-HG/terminator-3'
d) 5'-plant-specific promoter/pro-vitamin E/terminator-3'
5 The invention also expressly relates to vectors which are capable
of expressing polypeptides with an HGD, MAAI or FAAH activity.
The sequences encoding these genes are preferably derived from
plants, cyanobacteria, mosses, fungi or algae. The sequences
encoding polypeptides of SEQ ID N0: 3, 5 and 18 are especially
10 preferred.
In this context, the coding pro-HG or pro-vitamin E sequence, and
the sequences for the expression of polypeptides with HGD, MAAI
or FAAH activity, may also be replaced by a coding sequence for a
15 fusion protein of transit peptide and the sequence in question.
Preferred examples encompass vectors and may comprise one of the
following expression constructs:
a) 5'-35S promoter/anti-MAAI/FAAH/OCS terminator-3'
b) 5'-35S promoter/anti-HGD/OCS terminator-3';
c) 5'-legumin B promoter/pro-HG/NOS terminator-3'
d) 5'-legumin B promoter/pro-vitamin E/NOS-terminater-3'
e) 5'-legumin B promoter/HGD/NOS terminator-3'
f) 5'-legumin B promoter/MAAI/NOS terminator-3'
g) 5'-legumin B promoter/FAAH/NOS terminator-3'
In this context, too, the coding pro-HG sequence or pro-vitamin E
sequence may also be replaced by a coding sequence for a fusion
protein of transit peptide and pro-HG or pro-vitamin E.
A cotransformation with more than one of the abovementioned
examples a.) to g.) may be required for the advantageous
processes according to the invention for optimizing vitamin E
biosynthesis. Furthermore, transformation with one or more
vectors, each of which comprises a combination of the
abovementioned constructs, may be advantageous. Preferred
examples encompass vectors comprising the following constructs:
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16
a) 5'-35S promoter/anti-MAAI/FAAH/OCS terminator/legumin B
promoter/pro-HG/NOS terminator-3';
b) 5'-35S promoter/anti-MAAI/FAAH/OCS terminator/legumin B
promoter/pro-vitamin E/NOS terminator-3';
c) 5'-35S promoter/anti-HGD/OCS terminator/legumin B promoter/
pro-vitamin E/NOS terminator-3';
d) 5'-35S promoter/pro-HG/OCS terminator/legumin B promoter/
pro-vitamin E/NOS terminator-3';
Constructs a) tc d) permit the simultaneous transformation of the
plant with pro-HG and/or pro-vitamin E and anti-HGD and/or anti-
MAAI/FAAH .
Using the above-cited recombination and cloning techniques, the
nucleic acid constructs can be cloned into suitable vectors which
make possible the amplification, for example in E. coli. Suitable
cloning vectors are, inter alia, pBR332, pUC series, Ml3mp series
and pACYC184. Especially suitable are binary vectors which are
capable of replicating both in E. coli and in agrobacteria.
The nucleic acid constructs according to the invention are
preferably inserted into suitable transformation vectors.
Suitable vectors are described, inter alia, in Methods in Plant
Molecular Biology and Biotechnology (CRC Press), Chapter 6/7,
pp. 71-119 (1993). They are preferably cloned into a vector such
as, for example, pBinl9, pBinAR, pPZP200 or pPTV, which is
suitable for transforming Agrobacterium tumefaciens. The
agrobacteria transformed with such a vector can then be used in
the known manner for transforming plants, in particular crop
plants such as, for example, oilseed rape, for example by bathing
scarified leaves or leaf sections in an agrobacterial solution
and subsequently culturing them in suitable media. The
transformation of plants by agrobacteria is known, inter alia,
from F.F. White, Vectors for Gene Transfer in Higher Plants; in
Transgenic Plants, Vol. 1, Engineering and Utilization, edited by
S.D. Kung and R. Wu, Academic Press, 1993, pp. 15 - 38.
Transgenic plants which comprise the above-described nucleic acid
constructs integrated can be regenerated from the transformed
cells of the scarified leaves or leaf sections in the known
manner.
The nucleic acid sequences present in the nucleic acid constructs
and vectors according to the invention can be linked functionally
to at least one genetic control sequence. Genetic control
CA 02422760 2003-03-17
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17
sequences ensure for example transcription and translation in
prokaryotic or eukaryotic organisms. The constructs according to
the invention preferably comprise, 5'-upstream of the coding
sequence in question, a promoter and 3'-downstream a terminator
sequence and, if appropriate, other customary regulatory
elements, in each case functionally linked to the coding
sequence. Functional linkage is to be understood as meaning, for
example, the sequential arrangement of promoter, coding sequence,
terminator and, if appropriate, further regulatory elements in
such a way that each of the regulatory elements can fulfill its
intended function upon expression of the coding sequence or the
antisense sequence. This does not necessarily require direct
linkage in the chemical sense. Genetic control sequences such as;
for example, enhancer sequences, can also exert their function
from other DNA molecules toward the target sequence.
Examples are sequences to which inductors or repressors bind,
thus regulating the expression of the nucleic acid. In addition
to these novel control sequences, or instead of these sequences,
the natural regulation of these sequences before the actual
structural genes may still be present and, if appropriate, may
have been modified genetically so that the natural regulation has
been switched off and expression of the genes has been increased.
However, the nucleic acid construct may also have a simpler
structure, that is to say no additional regulatory signals are
inserted before the abovementioned genes, and the natural
promoter with its regulation is not removed. Instead, the natural
control sequence is mutated in such a way that regulation no
longer takes place and gene expression is enhanced. These
modified promoters may also be placed before the natural genes by
themselves in order to increase the activity.
Moreover, the nucleic acid construct may advantageously comprise
one or more enhancer sequences linked functionally to the
promoter, and these make possible an increased expression of the
nucleic acid sequence. At the 3' end of the DNA sequences, too,
additional advantageous sequences may be inserted, such as
further regulatory elements or terminators. The genes mentioned
hereinabove may be present in the gene construct in the form of
one or more copies.
Additional sequences which are preferred for functional linkage,
but not limited thereto, are further targeting sequences which
differ from the transit-peptide-encoding sequences and which
ensure subcellular localization in the apoplasts, in the vacuole,
in plastids, in the mitochondrion, in the endoplasmatic reticulum
(ER), in the nucleus, in eleoplasts or other compartments; and
0817/00017
CA 02422760 2003-03-17
18
translation enhancers such as the tobacco mosaic virus 5' leader
sequence (Gallie et al., Nucl. Acids Res. 15 (1987), 8693-8711),
and the like.
Control sequences are furthermore to be understood as those
sequences which make possible homologous or heterologous
recombination and/or insertion into the genome of a host
organism, or which permit the removal from the genome. In the
case of homologous recombination, the endogenous gene may be
inactivated fully, for example. Furthermore, it may be exchanged
for a synthetic gene with increased and modified activity.
Methods such as the cre/lox technology permit tissue-specific, in
some cases inducible, removal of the target gene from the genome
of the host organism (Sauer B. Methods. 1998; 14(4):381-92). This
involves adding certain flanking sequences (lox sequences) to the
target gene, which later make possible removal by means of cre
recombinase.
Various control sequences are suitable, depending on the host
organism or starting organism described in greater detail
hereinbelow which is transformed into a genetically modified or
transgenic organism by introducing the nucleic acid constructs.
Advantageous control sequences for the nucleic acid constructs
according to the invention, for the vectors according to the
invention, for the process according to the invention for the
preparation of vitamin E and for the genetically modified
organisms described hereinbelow are present, for example, in
promoters such as cos, tac, trp, tet, lpp, lac, lpp-lac, lacIq,
T7, T5, T3, gal, trc, ara, SP6, 1-PR or in the 1-PL promoter, all
of which are advantageously used Gram-negative bacteria.
Further advantageous control sequences are present, for example,
in the Gram-positive promoters amy and SP02, in the yeast or
fungal promoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH
or in the plant promoters CaMV/35S [Franck et al., Cell 21(1980)
285-294], PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)], SSU,
OCS, LEB4, USP, STLS1, B33, NOS; FBPaseP (WO 98/18940) or in the
ubiquitin or phaseolin promoter.
A preferred promoter for the nucleic acid constructs is, in
principle, any promoter which is capable of governing the
expression of genes, in particular foreign genes, in plants. A
promoter which is preferably used is, in particular, a plant
promoter or a promoter derived from a plant virus. Especially
preferred is the cauliflower mosaic virus CaMV 35S promoter
(Franck et al., Cell 21 (1980), 285 - 294). As is known, this
CA 02422760 2003-03-17
19
promoter comprises various recognition sequences for
transcriptional effectors which, in their totality, lead to
permanent and constitutive expression of the gene which has been
inserted (Benfey et al., EMBO J. 8 (1989). 2195-2202). A further
example of a suitable promoter is the Iegumin B promoter
(accession No. X03677).
The nucleic acid constructs may also comprise a chemically
inducible promoter by means of which expression of the exogenous
gene in the plant can be governed at a particular point in time.
Such promoters, such as, for example, the PRP1 promoter (Ward et
al.,-Plant. Mol. Biol. 22 (1993), 361-366), a ._ _
salicylic-acid-inducible promoter (WO 95/19443), a
benzenesulfonamide-inducible promoter (EP-A-0388186), a
tetraclclin-inducible promoter (Gatz et al., (1992) Plant J. 2,
397404), an abscisic-acid-inducibl,e promoter (EP-A 335528) or an
ethanol- or cyclohexanone-inducible promoter (WO 93/21334) may
also be used.
Furthermore, particularly preferred promoters are those which
ensure expression in tissues or plant parts in which the
biosynthesis of vitamin E or its precursors takes place or in
which the products are advantageously accumulated. Promoters
which must be mentioned in particular are those for the entire
plant owing to constitutive expression, such as, for example, the
CaMV promoter, the Agrobacterium OCS promoter (octopine
synthase), the Agrobacterium NOS promoter (nopaline synthase),
the ubiquitin promoter, promoters of vacuolar ATPase subunits, or
the promoter of a prolin-rich protein from wheat (WO 91/13991).
Promoters which must be mentioned in particular are those which
ensure leaf-specific expression. Promoters which must be
mentioned are the potato cytosolic FBPase promoter (WO 97/05900),
the Rubisco (ribulose-1,5-bisphosphate carboxylase) SSU (small
subunit) promoter, or the potato ST-LSI promoter (Stockhaus et
al., EMBO J. 8 (1989), 244 - 245). Examples of seed-specific
promoters are the phaseolin promoter (US 5504200), the USP
promoter (Baumlein, H. et al., Mol. Gen. Genet. (1991) 225 (3),
459 - 467) or the LEB4 promoter (Fiedler, U. et al.,
Biotechnology (NY) (1995), 13 (10) 1090) together with the LEB4
signal peptide.
Examples of other suitable promoters are specific promoters for
tubers, storage roots or roots, such as, for example, the patatin
promoter class I (B33), the potato cathepsin D inhibitor
promoter, the starch synthase (GBSS1) promoter or the sporamin
promoter, fruit-specific promoters such as, for example, the
tomato fruit-specific promoter (EP-A 409625),
CA 02422760 2003-03-17
fruit-maturation-specific promoters such as, for example, the
tomato fruit-maturation-specific promoter (WO 94/21794),
flower-specific promoters such as, for example, the phytoene
synthase promoter (WO 92/16635) or the promoter of the P-rr gene
5 (WO 98/22593) or specific plastid or chromoplast promoters such
as, for example, the RNA polymerase promoter (WO 97/06250) or
else the Glycine max phosphoribosyl pyrophosphate
amidotransferase promoter (see also Genbank Accession Number
U87999) or another node-specific promoter such as in EP-A 249676.
In principle, all natural promoters together with their
regulatory sequences such as those mentioned above can be used
for the process according to the invention. In addition,
synthetic promoters can also be used advantageously.
Polyadenylation signals which are-suitable as control sequences
are plant polyadenylation signals, preferably those which
essentially correspond to Agrobacterium tumefaciens T-DNA
polyadenylation signals, in particular to gene 3 of the T-DNA
(octopine synthase) of the Ti plasmid pTi.ACHS (Gielen et al.,
EMBO J. 3 (1984), 835 et seq.) or functional equivalents thereof.
Examples of particularly suitable terminator sequences are the
OCS (octopine synthase) terminator and the NOS (nopaline
synthase) terminator.
A nucleic acid construct is generated, for example, by fusing a
suitable promoter to a suitable anti-HGD, anti-MAAI/FAAH, pro-HG,
pro-vitamin E, HGD, MAAI or FAAH nucleotide sequence, if
appropriate a sequence encoding a transit peptide, preferably a
chloroplast-specific transit peptide, which sequence is
preferably arranged between the promoter and the nucleotide
sequence in question, and a terminator or polyadenylation signal.
To do this, customary recombination and cloning techniques are
used as they are described, for example, in T. Maniatis,
E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
(1989) and in T.J. Silhavy, M.L. Berman and L.W. Enquist,
Experiments with Gene Fusions, Cold Spring Harbor Laboratory,
Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al.,
Current Protocols in Molecular Biology, Greene Publishing Assoc.
and Wiley Interscience (1987).
As already mentioned, it is also possible to use nucleic acid
constructs whose DNA sequence encodes a pro-HG, pro-vitamin E,
HGD, MAAI or FAAH fusion protein, a portion of the fusion protein
being a transit peptide which governs the translocation of the
polypeptide. The following may be mentioned by way of example:
CA 02422760 2003-03-17
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21
chloroplast-specific transit peptides which are eliminated
enzymatically after translocation into the chloroplasts.
The pro-HG, pro-vitamin E, HGD, MAAI or FAAH nucleotide sequences
are preferably linked functionally to the coding sequence of a
plant organell-specific transit peptide. The transit peptide
preferably has specificity for individual cell compartments of
the plant, for example the plastids, such as, for example, the
chloroplasts, chromoplasts and/or leukoplasts. The transit
peptide guides the polypeptides which have been expressed to the
desired target in the plant and, once the target is reached, is
eliminated, preferably proteolytically. In the expression
construct according to the invention, the coding transit peptide
sequence is preferably located 5'-upstream of the coding pro-HG,
pro-vitamin E, HGD, MAAT or FAAH sequence. A transit peptide
which must be mentioned in particular is the transit peptide
which is derived from the plastid Nicotiana tabacum transketolase
(TK) or a functional equivalent of this transit peptide (for
example the transit peptide of the RubisCO small subunit, or of
ferredoxin:NADP oxidoreductase or else isopentenyl. pyrophosphate
isomerase-2).
The invention furthermore relates to transgenic organisms
transformed with at least one nucleic acid construct according to
the invention or a vector according to the invention, and to
cells, cell cultures, tissues, parts - such as, for example,
leaves, roots and the like in the case of plant organisms - or
propagation material derived from such organisms.
Organisms, starting organisms or host organisms are to be
understood as meaning prokaryotic or eukaryotic organisms such
as, for example, microorganisms or plant organisms. Preferred
microorganisms are bacteria, yeasts, algae or fungi.
Preferred bacteria are bacteria of the genus Escherichia,
Erwinia, Agrobacterium, Flavobacterium, Alcaligenes or
cyanobacteria, for example, of the genus Synechocystis.
Preferred microorganisms are, above all, those which are capable
of infecting plants and thus of transferring the constructs
according to the invention. Preferred microorganisms are those
from among the genus Agrobacterium and, in particular, the
species Agrobacterium tumefaciens.
Preferred yeasts are Candida, Saccharomyces, Hansenula or Pichia.
0817/00017 CA 02422760 2003-03-17
22
Plant organisms are, for the purposes of the invention,
monocotyledonous and dicotyledonous plants. The trasngenic plants
according to the invention are selected in particular from among
monocotyledonous crop plants such as, for example, cereals such
5 as wheat, barley, sorghum and millet, rye, triticale, maize, rice
or oats, and sugar cane. The transgenic plants according to the
invention are furthermore selected in particular from among
dicotyledonous crop plants such as, for example,
10 Brassicaceae such as oilseed rape, cress, Arabidopsis, cabbages
or canola,
Leguminosae such as soybean, alfalfa, pea, bean plants or peanut
Solanaceae such as potato, tobacco, tomato, aubergine or bell
pepper,
15 Asteraceae such as sunflower, Tagetes, lettuce or calendula,
Cucurbitaceae such as melon, pumpkin or zucchini,
and also linseed, cotton, hemp, flax, red pepper, carrot, sugar
beet and the various tree, nut and grapevine species.
20 Especially preferred are Arabodopsis thaliana, Nicotiana tabacum,
Tagetes erecta, Calendula vulgaris and all genera and species
which are suitable for the production of oils, such as oil crops
(such as, for example, oilseed rape), nut species, soybean,
sunflower, pumpkin and peanut.
Plant organisms for the purposes of the invention are,
furthermore, further photosynthetically active organisms, or
organisms which are capable of synthesizing vitamin E, such as,
for example, algae or cyanobacteria, and also mosses.
Preferred algae are green algase, such as, for example, algae of
the genus Haematococcus, Phaedactylum tricornatum, Volvox or
Dunaliella.
The transfer of foreign genes into the genome of an organism, for
example a plant, is termed transformation. It exploits the
above-described methods of transforming and regenerating plants
from plant tissues or plant cells for transient or stable
transformation. Suitable methods are protoplast transformation by
polyethylene glycol-induced DNA uptake, the biolistic method
using the gene gun, the particle bombardment method,
electroporation, incubation of dry embryos in DNA-containing
solution, microinjection and agrobacterium-mediated gene
transfer. The abovementioned methods are described, for example,
in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic
Plants, Vol. 1, Engineering and Utilization, edited by S.D. Kung
and R. Wu, Academic Press (1993), 128 - 143 and in Potrykus,
0$17/0001.7 CA 02422760 2003-03-17
23
Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 20S -
225). The construct to be expressed is preferably cloned into a
vector which is suitable for transforming Agrobacterium
tumefaciens for example pBinl9 (Bevan et al., Nucl. Acids Res. 12
(1984), 8711).
The expression efficacy of the recombinantly expressed nucleic
acids can be determined, for example, in vitro by shoot-meristem
propagation. In addition, changes in the nature and level of the
expression of the pro-HG or pro-vitamin E genes and their effect
on vitamin E biosynthesis performance, can be tested on test
plants in greenhouse experiments.
The invention furthermore relates to transgenic organisms as
described above whose vitamin E production is improved in
comparison with the untransformed wild type.
In accordance with the invention are furthermore cells, cell
cultures, parts - such as, for example, roots, leaves etc. in the
case of transgenic plant organisms -, transgenic propagation
material, seeds or fruit derived from the above-described
transgenic organisms.
Improved vitamin E production means for the purposes of the
present invention for example the artificially acquired ability
of an increased biosynthesis performance of at least one compound
from the group of the tocopherols and tocotrienols in the
transgenic organism in comparison with the non-genetically
modified starting organism for the duration of at least one plant
generation. Preferably, the vitamin E production in the
transgenic organism in comparison with the non-genetically
modified starting organism, is increased by 10~, especially
preferably by 50~, very especially preferably by 100. The term
improved may also mean an advantageously modified qualitative
composition of the vitamin E mixture.
The biosynthesis site of vitamin E is, generally, the leaf
tissue, but also the seed, so that leaf-specific or seed-specific
expression of, in particular, pro-HG and pro-vitamin E sequences
and, if appropriate, anti-HGD and/or anti-MAAI/FAAH sequences is
meaningful. However, it is obvious that vitamin E biosynthesis
need not be restricted to the seed, but can also take place in a
tissue-specific manner in all remaining parts of the plant. In
addition, constitutive expression of the exogenous gene is
advantageous. On the other hand, inducible expression may also be
desirable.
~817/~~~17
CA 02422760 2003-03-17
24
Finally, the invention furthermore relates to a process for the
production of vitamin E, which comprises isolating the desired
vitamin E in a manner known per se from a culture of a plant
organism which has been transformed in accordance with the
invention.
Genetically modified plants according to the invention with an
increased vitamin E content which can be consumed by humans and
animals can also be used as foodstuffs or feed, for example
directly or following processing, which is known per se.
The invention furthermore relates to the use of polypep.tides _
which encode an HGD, MAAI or FAAH, of the genes and cDNAs on
which they are based, and/or of the nucleic acid constructs
according to the invention, vectors according to the invention or
organisms according to the invention which are derived from them
for producing antibodies, protein-binding or DNA-binding factors.
The biosynthetic pathway of the HGD-MAAI-FAAH catabolic pathway
offers target enzymes for the development of inhibitors.
Therefore, the invention also relates to the use of polypeptides
which encode an HGD, MAAI or FAAH, of the genes and cDNAs on
which they are based, and/or of the nucleic acid constructs
according to the invention, vectors according to the invention or
organisms according to the invention which are derived from them
as target for finding inhibitors of HGD, MA.AI or FAAH.
To be able to find efficient HGD, MAAI or FAAH inhibitors, it is
necessary to provide suitable assay systems with which
inhibitor-enzyme binding studies can be carried out. To this end,
for example, the complete cDNA sequence of HGD, MAAI or FAAH is
cloned into an expression vector (for example pQE, Qiagen) and
overexpressed in E. call. The HGD, MAA,I or FAAH proteins are
particularly suitable for finding HGD-, MAAI- or FAAH-specific
inhibitors.
Accordingly, the invention relates to a process for finding
inhibitors of HGD, MAAI or FAAH using the abovementioned
polypeptides, nucleic acids, vectors or transgenic organisms,
which comprises measuring the enzymatic activity of HGD, MAAI or
FAAH in the presence of a chemical compound and, if the enzymatic
activity is reduced in comparison with the unhibited activity,
the chemical compound constitutes an inhibitor. To this end, HGD,
MAAI or FAAH can be employed, for example, in an enzyme assay in
which the activity of HGD, MAAI or FAAH is determined in the
presence and absence of the active ingredient to be assayed.
Qualitative and quantitative findings on the inhibitory behavior
CA 02422760 2003-03-17
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of the active ingredient to be assayed can be deduced by
comparing the two activity determinations. A multiplicity of
chemical compounds can be tested in a simple and rapid fashion
for herbicidal properties with the aid of the assay system
5 according to the invention. The method allows reproducibly to
select, from a large number of substances, specifically those
which are very potent in order to subject these substances
subsequently to further, in-depth tests with which the skilled
worker is familiar.
The inhibitors of HGD, MAAI or FAAH are suitable for functionally
increasing vitamin E biosynthesis similarly to the
above-described anti-HGD and/or anti-MAAI/FAAH nucleic acid
sequences. The invention therefore furthermore relates to
processes for improving the vitamin E production using inhibitors
of HGD, MAAI or FAAH. The improved production of vitamin E can
have a positive effect on the plant since these compounds have an
important function in the protection from harmful environmental
factors (sun rays, free-radical oxygen). An increased vitamin E
production can thus act as growth promoter. The invention
therefore furthermore relates to the use of inhibitors of HGD,
MAAI or FAAH, obtainable by the above-described process, as
growth regulators.
Sequences
SEQ ID NO. 1; Arabidopsis thaliana homogentisate 1,2-dioxygenase
(HGD) gene
SEQ ID N0. 2: Arabidopsis thaliana homogentisate 1,2-dioxygenase
(HGD) cDNA
SEQ ID N0. 3: Arabidopsis thaliana homogentisate 1,2-dioxygenase
(HGD) polypeptide
SEQ ID NO. 4: Arabidopsis thaliana fumaryl acetoacetate
hydrolase (FAAH) cDNA
SEQ ID N0. 5: Arabidopsis thaliana fumaryl acetoacetate
hydrolase (FAAH) polypeptide
SEQ ID N0. 6: Arabidopsis thaliana maleyl-acetoacetate isomerase
(MAAI) gene
SEQ ID N0. 7: TyrA gene encoding a bifunctional chorismate
mutase/ prephenate dehydrogenase
SEQ ID NO. 8: TyrA polypeptide encoding a bifunctional
chorismate mutase/ prephenate dehydrogenase
SEQ ID N0. 9: Arabidopsis thaliana fumaryl acetoacetate
hydrolase (FAAH) gene
SEQ ID NO. 10: Arabidopsis thaliana hydroxyphenyl-pyruvate
dioxygenase (HPPD) cDNA
CA 02422760 2003-03-17
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26
SEQ ID N0. 11: Arabidopsis thaliana hydroxyphenyl-pyruvate
dioxygenase (HPPD) polypeptide
SEQ ID N0. 12: Brassica napus homogentisate 1,2-dioxygenase (HGD)
cDNA fragment
SEQ ID N0. 13: Synechocystis PCC6803 homogentisate
phythyltransferase cDNA
SEQ ID N0. 14: Synechocystis PCC6803 homogentisate
phythyltransferase polypeptide
SEQ ID NO. 15: artificial codon usage optimized cDNA encoding
Streptomyces avermitilis hydroxyphenyl-pyruvate
dioxygenase (HPPDop)
SEQ ID NO. 16: Stre_ptomyces avermitilis hydroxyphenyl-pyruvate
dioxygenase polypeptide
SEQ ID NO. 17: Arabidopsis thaliana maleyl-acetoacetate isomerase
( MAAI ) c DILTA
SEQ ID N0. 18: Arabidopsis thaliana maleyl-acetoacetate isomerase
(MAAI) polypeptide
SEQ ID N0. 19: Arabidopsis thaliana 'y-tocopherol
methyltransferase cDNA
SEQ ID NO. 20: Arabidopsis thaliana y-tocopherol
methyltransferase polypeptide
SEQ ID NO. 21: Synechocystis PCC6803
3-methyl-6-phytylhydroquinone methyltransferase
cDNA
SEQ ID NO. 22: Synechocystis PCC6803
3-methyl-6-phytylhydroquinone methyltransferase
polypeptide
SEQ ID N0. 23: Nicotiana tabacum geranylgeranyl pyrophosphate
oxidoreductase cDNA
SEQ ID NO. 24: Nicotiana tabacum geranylgeranyl pyrophosphate
oxidoreductase polypeptide
SEQ ID NO. 25: Primer (5'-HGD Brassica napus)
5'-GTCGACGGNCCNATNGGNGCNAANGG-3'
SEQ ID N0. 26: Primer (3'-NOS terminator)
5'-AAGCTTCCGATCTAGTAACATAGA-3'
SEQ ID NO. 27: Primer (5'-35S promoter)
5'-ATTCTAGACATGGAGTCAAAGATTCAAATAGA-3'
SEQ ID NO. 28: Primer (3'-OCS terminator)
5'-ATTCTAGAGGACAATCAGTAAATTGAACGGAG-3'
SEQ ID N0. 29: Primer (5'-MAAI A.thaliana)
5'-atgtcgacATGTCTTATGTTACCGAT-3'
SEQ ID NO. 30: Primer (3'-MAAI A.thaliana)
5'-atggatccCTGGTTCATATGATACA-3'
SEQ ID N0. 31: Primer (5'-FAAH A.thaliana)
5'-atgtcgacGGAAACTCTGAACCATAT-3'
SEQ ID NO. 32: Primer (3'-FAAH A.thaliana)
5'-atggtaccGAATGTGATGCCTAAGT-3'
7 / 00017 CA 02422760 2003-03-17
27
SEQ ID NO. 33: Primer (3'-HGD Brassica napus)
5'-GGTACCTCRAACATRAANGCCATNGTNCC-3'
SEQ ID N0. 34: Primer (5'-legumin promoter)
5'-GAATTCGATCTGTCGTCTCAAACTC-3'
SEQ ID N0. 35: Primer (3'-legumin promoter}
5'-GGTACCGTGATAGTAAACAACTAATG-3'
SEQ ID N0. 36: Primer (5'-transit peptide}
5'-ATGGTACCTTTTTTGCATAAACTTATCTTCATAG-3'
SEQ ID N0. 37: Primer (3'-transit peptide}
5'-ATGTCGACCCGGGATCCAGGGCCCTGATGGGTCCCATTTTCCC-3'
SEQ ID N0. 38: Primer (5'-NOS terminator)
5'-GTCGACGAATTTCCCCGAATCGTTC-3'
SEQ ID N0. 39: Primer (3'-NOS terminator II)
5'-AAGCTTCCGATCTAGTAACATAGA-3'
SEQ ID NO. 40: Primer (5'-legumin promoter II)
5'-AAGCTTGATCTGTCGTCTCAAACTC-3'
SEQ ID NO. 41: Arabidopsis thaliana maleyl-acetoacetate isomerase
(MAAI) gene (fragment)
SEQ ID N0. 42: Arabidopsis thaliana fumaryl acetoacetate
hydrolase (FAAH) gene (fragment)
SEQ ID NO. 43: Primer (5'-35S promoter)
5'-ATGAATTCCATGGAGTCAAAGATTCAAATAGA-3'
SEQ ID N0. 44: Primer (3'-OCS terminator)
5'-ATGAATTCGGACAATCAGTAAATTGAACGGAG-3'
Examples
The invention is illustrated in greater detail in the use
examples which follow with reference to the appended figures.
Abbreviations with the following meanings are used:
A 35S promoter B = HGD in antisense orientation
=
C OCS terminator D = legumin B promoter
=
E FNR transit peptide F = HPPDop
=
(HPPD with optimized codon usage)
G NOS terminator H = MAAI in antisense orientation
=
I FAAH
= in
antisense
orientation
The direction of arrows in the figures indicates in each case the
direction in which the genes in question are read. In the
figures:
Figure 1 shows a schematic representation of the vitamin E
biosynthetic pathway in plants;
CA 02422760 2003-03-17
28
Figure 2 shows construction schemes of the anti-HGD-coding
plasmids pBinARHGDanti (I) and pCRScriptHGDanti (II);
Figure 3 shows construction schemes of the HPPDop-coding plasmids
pUCI9HPPDop (III) and pCRScriptHPPDop (IV);
Figure 4 shows construction schemes of the transformation vectors
pPTVHGDanti (V) and of the bifunctional transformation
vector pPTV HPPDop HGD anti (VI), which expresses HPPDop
in the seeds of transformed plants while simultaneously
suppressing the expression of the endogenous HGD;
Figure 5 shows a construction scheirae of the transformation vector
pPZP200HPPDop (VII).
Figure 6 shows construction schemes of the transformation vectors
pGEMT MAAI1 anti (VIII) and pBinAR MAAT1 anti (IX);
Figure 7 shows construction schemes of the transformation vectors
pCR-Script MAAI1 anti (X) and pZPNBN MAAI1 anti (XI);
Figure 8 shows the construction scheme of the transformation
vector pGEMT FAAH anti (XII);
Figure 9 shows construction schemes of the transformation vectors
pBinAR FAAH anti (XIII) and pZPNBN FAAH anti (XIV).
General methods:
The chemical synthesis of oligonucleotides can be carried out for
example in the known manner by the phosphoamidite method (Voet,
Voet, 2nd Edition, Wiley Press New York, pp. 896-897). The
cloning steps carried out within the present invention such as,
for example, restriction cleavages, agarose gel electrophoresis,
purification of the DNA fragments, transfer of nucleic acids to
nitrocellulose and nylon membranes, linking DNA fragments,
transformation of E. coli cells, bacterial cultures, phage
replication and sequence analysis of recombinant DNA, were
carried out as described by Sambrook et al. (1989) Cold Spring
Harbor Laboratory .Press; ISBN 0-87969-309-6. Recombinant DNA
molecules were sequenced using a Licor laser fluorescence DNA
sequencer (supplied by MWG Biotech, Ebersbach) using the method
of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977),
5463-5467).
081~~~~~~1~~ CA 02422760 2003-03-17
29
Example 1:
Cloning a hydroxyphenyl-pyruvate dioxygenase (HPPD) with a DNA
sequence optimized for expression in Brassica napes
The amino acid sequence of the Streptomyces avermitilis
hydroxyphenyl-pyruvate dioxygenase (HPPD) (Accession No. U11864,
SEQ ID N0:16) was backtranslated to a DNA sequence taking into
consideration the codon usage in Brassica napes (oilseed rape).
The codon usage was determined by means of the database
http://www.dna.affrc.go.jp/ -nakamura/index.html. The derived
sequence was synthesized by ligating overlapping oligonucleotides
followed by PCR amplification, attaching SalI cleavage sites
w (Rouwendal, GJA; et al, (1997) PMB 33: 989-999) (SEQ ID N0:15).
The correctness of the sequence of the synthetic gene was
verified by sequencing. The synthetic gene was cloned to the
vector pBluescript II SK+ (Stratagene). (This codon-optimized
sequence is subsequently also termed HPPDop.)
Example 2:
Cloning a Brassica napes homogentisate dioxygenase (HGD)
a) Isolating total RNA from Brassica napes flowers
Open flowers were harvested from Brassica napes var. Westar and
frozen in liquid nitrogen. The material was subsequently reduced
to a powder in a mortar and taken up in Z6 buffer (8 M
guanidinium hydrochloride, 20 mM MES, 20 mM EDTA, brought to pH
7.0 with NaOH; immediately prior to use, 400 ml of
mercaptoethanol/100 ml of buffer were added). The suspension was
then transferred into reaction vessels and extracted by shaking
with one volume of phenol/chloroform/isoamyl alcohol 25:24:1.
After centrifugation for 10 minutes at 15,000 rpm, the
supernatant was transferred into a new reaction vessel and the
RNA was precipitated with 1/20 volume of 1N acetic acid and 0.7
volume of (absolute) ethanol. After a further centrifugation
step, the pellet was first washed in 3M sodium acetate solution
and, after another centrifugation step, in 70~ strength ethanol.
The pellet was subsequently dissolved in DEPC
(diethylpyrocarbonate) water and the RNA concentration determined
photometrically.
b) Preparation of cDNA from total RNA from Brassica napes
flowers
20 mg of total RNA were first treated with 3.3 ml of 3M sodium
acetate solution and 2 ml of 1M magnesium sulfate solution and
the mixture was made up to an end volume of 10 ml with DEPC
' 0x17/00017 CA 02422760 2003-03-17
water. 1 ml of RNase-free DNase (Boehringer Mannheim) was added,
and the mixture was incubated for 45 minutes at 37 degrees. After
the enzyme had been removed by extracting by shaking with
phenol/chloroform/isoamyl alcohol, the RNA was precipitated with
5 ethanol and the pellet was taken up in 100 ml of DEPC water.
2.5 mg of RNA from this solution were transcribed into cDNA by
means of a cDNA kit (Gibco BRL) following the manufacturer's
instructions.
10 c) PCR amplification of a part-fragment of the 9rassica napus
HGD
Oligonucleotides which had been provided with an SaII restriction
cleavage site at the 5' end and with an Asp718 restriction
15 cleavage site at the 3' end were derived for a PCR by aligning
the DNA sequences of the known homogentisate dioxygenases (HGDs)
from Arabidopsis thaliana (Accession No. U80668), Homo sapiens
(Accession No. U63008) and Mus musculus (Accession No. U58988).
The oligonucleotide at the 5' end comprises the sequence:
5'-GTCGACGGNCCNATNGGNGCNAANGG-3' (SEQ ID N0:25),
starting with base 661 of the Arabidopsis gene. The
oligonucleotide at the 3' end comprises the sequence:
5'-GGTACCTCRAACATRAANGCCATNGTNCC-3' (SEQ ID N0:33),
starting with base 1223 of the Arabidopsis gene, N in each case
denoting inosine and R denoting the incorporation of A or G into
the oligonucleotide.
The PCR reaction was carried out with TAKARA Taq polymerase
following the manufacturer's instructions. 0.3 mg of the cDNA was
employed as template. The PCR program was:
~ 1 cycle at: 94°C (1 min)
5 cycles at: 94°C (4 sec), 50°C (30 sec), 72°C (1 min)
5 cycles at: 94°C (4 sec), 48°C (30 sec), 72°C (1 min)
25 cycles at: 94°C (4 sec), 46 degrees (30 sec), 72 degrees
(1 min)
1 cycle at: 72 degrees (30 min)
The fragment was purified by NucleoSpin Extract (Macherey and
Nagel) and cloned into vector pGEMT (Promega) following the
manufacturer's instructions. The correctness of the fragment was
verified by sequencing.
. , 0817/0001,7 CA 02422760 2003-03-17
31
Example 3: Generation of a plant transformation construct for
overexpressing the HPPD with optimized DNA sequence (HPPDop) and
eliminating HGD
To generate plants which express HPPDop in seeds and in which the
expression of the endogenous HGD is suppressed by antisense
technology, a binary vector which contains both gene sequences
was constructed (Figure 4, construct VI).
a) Generation of an HPPDop nucleic acid construct
To this end, the components of the cassette for expressing
HPPDop, composed of the legumin B promoter (Accession No.
X03677),- the spi~zach ferredoxin:NADP+ oxidoreductase transit
peptide (FNR; Jansen, T, et al (1988) Current Genetics 13,
517-522) and the NOS terminator (present in pBI101 Accession No.
U12668) were first provided with the necessary restriction
cleavage sites using PCR.
The legumin promoter was amplified from plasmid.plePOCS
(B~umlein, H, et al. (1986) Plant J. 24, 233-239) with the
upstream oligonucleotide:
5'-GAATTCGATCTGTCGTCTCAAACTC-3' (SEQ ID NO: 34)
40
and the downstream oligonucleotide:
5'-GGTACCGTGATAGTAAACAACTAATG-3' (SEQ ID N0: 35)
by means of PCR and cloned into vector PCR-Script (Stratagene)
following the manufacturer's instructions.
The transit peptide was amplified with plasmid pSK-FNR (Andrea
Babette Regierer "Molekulargenetische Ansatze zur Veranderung der
Phosphat-Nutzungseffizienz von hoheren Pflanzen" [Molecular
genetic approaches for modifying the phosphate utilization
efficiency of higher plants], P+H 4Vissenschaftlicher Verlag,
Berlin 1998 ISBN: 3-9805474-9-3) by means of PCR using the 5'
oligonucleotide:
5'-ATGGTACCTTTTTTGCATAAACTTATCTTCATAG-3' (SEQ ID NO: 36)
and the 3' oligonucleotide:
5'-ATGTCGACCCGGGATCCAGGGCCCTGATGGGTCCCATTTTCCC-3' (SEQ ID N0: 37)
CA 02422760 2003-03-17
32
The NOS terminator was amplified from plasmid pBI101 (Jefferson,
R.A., et al (1987) EMBO J. 6 (13), 3901-3907) by means of PCR
using the 5' oligonucleotide:
5 5'-GTCGACGAATTTCCCCGAATCGTTC-3' (SEQ ID N0: 38)
and the 3' oligonucleotide
5'-AAGCTTCCGATCTAGTAACATAGA-3' (SEQ ID N0: 26)
The amplicon was cloned in each case into vector pCR-Script
(Stratagene) following the manufacturer's instructions.
For the nucleic acid constructs, the NOS terminator was first
recloned as SalI/HindIII fragment into a suitably cut pUCl9
vector (Yanisch-Perron, C., et al (1985) Gene 33, 103-119). The
transit peptide was subsequently introduced into this plasmid as
Asp718/SalI fragment. The legumin promoter was then cloned in as
EcoRI/Asp718 fragment. The gene HPPDop was introduced into this
construct as SalI fragment (Figure 3, construct III).
The finished cassette in pUCI9 was used as template for a PCR,
using the oligonucleotide:
5'-AAGCTTGATCTGTCGTCTCAAACTC-3' (SEQ ID NO: 40)
for the legumin promoter and the oligonucleotide:
5'-AAGCTTCCGATCTAGTAACATAGA-3' (SEQ ID NO: 39)
35
for the NOS terminator. The amplicon was cloned into pCR-Script
and termed pCR-ScriptHPPDop (Figure 3, construct IV).
d) Generation of an antiHGD nucleic acid construct
To switch off HGD by means of antisense technology, the gene
fragment was cloned as SalI/Asp718 fragment into vector pBinAR
(Hofgen, R. and Willmitzer, L., (1990) Plant Sci. 66: 221-230),
in which the 35S promoter and the OCS terminator are present
(Figure 2, construct I). The construct acted as template for a
PCR reaction with the oligonucleotide:
5'-ATTCTAGACATGGAGTCAAAGATTCAAATAGA-3' (SEQ ID N0: 27),
which is specific for the 35S promoter sequence;
CA 02422760 2003-03-17
33
and the oligonucleotide:
5'-ATTCTAGAGGACAATCAGTAAATTGAACGGAG-3' (SEQ ID N0: 28),
which is specific fox the OCS terminator sequence.
The amplicon was cloned into vector pCR-Script (Stratagene) and
termed pCRScriptHGDanti (Figure 2, construct II).
c) Preparation of the binary vector
To construct a binary vector for transforming oilseed rape, the
construct HGDanti from pCRScriptHGDanti was first cloned as XbaI
fragment into vector pPTV (Becker, D.,(1992) PMB 20, 1195-1197)
(Figure 4, construct V). The construct LegHPPDop from
pCRScriptHPPDop was inserted into this plasmid as-HindIII
fragment. This plasmid was termed pPTVHPPDopHGDanti (Figure 4,
construct VI).
Example 4:
Generation of constructs for the cotransformation for
overexpressing HPPDop and switching off HGD in Brassica napus
plants
To cotransform plants with HPPDop and antiHGD, the construct
legumin B promoter/transit peptide/HPPDop/NOS was excised from
vector pCRScriptHPPDop (Figure 3, construct IV) as HindIII
fragment and inserted into the correspondingly cut vector
pPZP200 (Hajdukiewicz, P., et al., (1994) PMB 25(6): 989-94)
(Figure 5, construct VII). This plasmid was used later for
cotransforming plants together with vector pPTVHGDanti (Figure 4,
construct V) of Example 3 c).
Example 5:
Cloning a genomic fragment of the Arabidopsis thaliana
maleyl-acetoacetate isomerase
a) Isolation of genomic DNA from A. thaliana leaves:
The extraction buffer used has the following composition:
~ 1 volume of DNA extraction buffer (0.35 M sorbitol, 0.1 M
Tris, 5 mM EDTA, pH 8.25 HC1)
~ 1 volume of nuclei lysis buffer (0.2 M Tris-HC1 pH 8.0, 50 rnM
EDTA, 2 M NaCl, 2~ hexadecyltrimethylammonium bromide (CTAB))
0817 ~ ~ ~ ~1,~ CA 02422760 2003-03-17
34
~ 0.4 volume of 5~ sodium sarcosyl
~ 0.38 g/100 ml sodium bisulfate
100 mg of leaf material of A tha3iana were harvested and frozen
in liquid nitrogen. The material was subsequently reduced to a
powder in a mortar and taken up in 750 ~1 of extraction buffer.
The mixture was heated for 20 minutes at 65°C and subsequently
extracted by shaking with one volume of chloroform/isoamyl
alcohol (24:1). After centrifugation for 10 minutes at 10,000 rpm
in a Heraeus pico-fuge, the supernatant was treated with one
volume of isopropanol, and the DNA thus precipitated was again _
pelleted for S minutes at 10,000 rpm. The pellet was washed in
70~ strength ethanol, dried for 10 minutes at room temperature
and subsequently dissolved in 100 ~,l of TE RNase buffer (10 mM
Tris HC1 pH 8.0, 1 mM EDTA pH 8.0, 100 mgfl RNase).
b) Cloning the gene for the Arabidopsis thaliana MA.AI
Using the protein sequence of mouse (Mus musculus) MAAI, the
A.thaliana MAAI gene was identified by means of BLAST search in
the NCBI database (http://www.ncbi.nlm.nih.gov/BLAST/) (Genbank
Acc.-No. AAC78520.1). The sequence is annotated in Genbank as
putative glutathione S-transferase. The corresponding DNA
sequence was determined by means of the ID numbers of the protein
sequence, and oligonucleotides were derived. An SalI restriction
cleavage site was added to the 5' end of each of the
oligonucleotides and a BamHI restriction cleavage site to the 3'
end of each of the nucleotides. The oligonucleotide at the 5' end
encompasses the sequence
5'-atgtcgacATGTCTTATGTTACCGAT-3' (SEQ ID N0: 29)
starting with base 37 of the cDNA, the first codon, the
oligonucleotide at the 3' end comprises the sequence
5'-atggatccCTGGTTCATATGATACA-3' (SEQ ID N0: 30)
starting with base pair 803 of the cDNA sequence. The PCR
reaction was carried out using Taq polymerase (manufacturer:
TaKaRa Shuzo Co., Ltd.). The composition of the mix was as
follows: 10 ~1 buffer (20 mM Tris-HC1 pH 8.0, 100 mM KC1, 0,1 mM
EDTA, 1 mM DTT, 0.5~ Tween20, 0.5$ Nonidet P-40, 50~ glycerol),
in each case 100 pmol of the two oligonucleotides, in each case
20 nM of dATP, dCTP, dGTP, dTTP, 2.5 units Taq polymerase, 1 ~g of
genomic DNA, distilled water to 100 ~tl. The PCR program was:
CA 02422760 2003-03-17
~ 5 cycles at: 94°C (4 sec), 52°C (30 sec), 72°C (1 min)
~ 5 cycles at: 94°C (4 sec), 50°C (30 sec), 72°C (1 min)
~ 25 cycles at: 94°C (4 sec), 48°C (30 sec), 72°C (1 min)
5 The amplified fragment (SEQ ID N0: 41) was purified by means of
Nucleo-Spin Extract (Macherey-Nagel) and cloned into the Promega
vector pGEMTeasy following the manufacturer's instructions
(Figure 6, construct VIII). The correctness of the fragment was
verified by sequencing. By means of the restriction cleavage
10 sites which had been added to the sequence by the primers, the
gene was cloned into the correspondingly cut vector pBinAR
(Hofgen, R. and Willmitzer, L., (1990) Plant Sci-. 66:,221-230) as
SalI/BamHI fragment (Figure 6, construct IX). This vector
contains the cauliflower mosaic virus 35S promoter and the OCS
15 termination sequence. The construct acted as template for a PCR
reaction with the oligonucleotide
5'-ATGAATTCCATGGAGTCAAAGATTCAAATAGA-3' (SEQ ID N0: 43),
20 which is specific for the 35S promoter sequence and the
oligonucleotide
5'-ATGAATTCGGACAATCAGTAAATTGAACGGAG-3' (SEQ ID N0: 44),
25 which is specific for the OCS terminator. An EcoRI recognition
sequence was added to both oligonucleotides. The PCR was carried
out using Pfu polymerase (manufacturer: Stratagene). The
composition of the mix was as follows: 10 ~1 of buffer (200 mM
Tris HCl pH 8.8, 20 mM MgS04, 100 mM KC1, 100 mM ammonium sulfate,
30 1~ Triton X-100, 1 g/1 nuclease-free BSA), in each case 100 pmol
of the two oligonucleotides, in each case 20 nM of dATP, dCTP,
dGTP, dTTP, 2.5 units Pfu polymerase, Z ng of plasmid DNA,
distilled water to 100 ~,1. The PCR program was:
35 ~ 5 cycles at: 94°C (4 sec), 52°C (30 sec), 72°C (2
min)
~ 5 cycles at: 94°C (4 sec), 50°C (30 sec), 72°C (2 min)
25 cycles at: 94°C (4 sec), 48°C (30 sec), 72°C (2 min)
The PCR fragment was purified by means of Nucleo-Spin Extract
(Macherey-Nagel) and cloned into vector pCR-Script (Stratagene)
(Figure 7, construct X).
Example 6: Generation of the binary vector
To construct a binary vector for transforming Arabidopsis and
oilseed rape, the construct from vector pCR-Script was cloned
into vector pZPNBN as EcoRI fragment. pZPNBN is a pPZP200
CA 02422760 2003-03-17
36
derivative (Hajdukiewicz, P., et al., (1994) PMB 25(6): 989-94),
into which a phosphinothricin resistance under the control of the
NOS promoter had been inserted before the NOS terminator. (Figure
7, construct XI)
Example 7: Cloning a genomic fragment of the Arabidopsis thaliana
fumaryl-acetoacetate isomerase
A BLAST search was carried out by means of the protein sequence
of the Emericella nidulans FAAH, and a protein sequence was
identified from A. thaliana which had 59~ homology. A. thaliana
FAAH has the Accession number AC002131. The DNA sequence was
determined by means of the ID number of the protein sequence, and
oligonucleotides were derived.
Z5
An SalI restriction cleavage site was added to the 5'
oligonucleotide and an Asp718 restriction cleavage site was added
to the 3' oligonucleotide. The oligonucleotide at the 5' end of
FAAH comprises the sequence
5'-atgtcgacGGAAACTCTGAACCATAT-3' (SEQ ID NO: 31)
starting with base 40258 of BAC F12F1, the oligonucleotide at the
3' end comprises the sequence:
5'-atggtaccGAATGTGATGCCTAAGT-3' (SEQ ID NO: 32)
starting with base pair 39653 of the BAC. The PCR reaction was
carried out with Taq polymerase (manufacturer: TaKaRa Shuzo Co.,
Ltd.). The composition of the mix was as follows: 10 ~l buffer
(20 mM Tris-HC1 pH 8.0, 100 mM KC1, 0.1 mM EDTA, 1 mM DTT, 0.5~
Tween20, 0.5~ Nonidet P-40, 50~ glycerol), in each case 100 pmol
of the two oligonucleotides, in each case 20 nM of dATP, dCTP,
dGTP, dTTP, 2.5 units Taq polymerase, 1 ~g genomic DNA, distilled
water to 100 ~1. The PCR program was:
~ 5 cycles at: 94°C (4 sec), 52°C (30 sec), 72°C (1 min)
~ 5 cycles at: 94°C (4 sec), 50°C (30 sec), 72°C (1 min)
~ 25 cycles at: 94°C (4 sec), 48°C (30 sec), 72°C (1 min)
The fragement (SEQ ID NO: 42) was purified by means of
Nucleo-Spin Extract (Macherey-Nagel) and cloned into the Promega
vector pGEMTeasy following the manufacturer's instructions
(Figure 8, construct XII).
7/00017 CA 02422760 2003-03-17
37
The correctness of the fragment was verified by sequencing. By
means of the restriction cleavage sites added to the sequence of
the primers, the gene was cloned as Sall/Asp718 fragment into the
correspondingly cut vector pBinAR (Hofgen, R. and Willmitzer, L.,
Plant Sci. 66: 221-230, 1990). This vector contains the
cauliflower mosaic virus 35S promoter and the OCS termination
sequence (Fig. 9, construct XIII).
To construct a binary vector for transforming Arabidopsis and
oilseed rape, the construct from vector pBinAr was cloned into
vector pZPNBN as EcoRI/HindIII fragment. pZPNBN is a pPZP200
derivative (Hajdukiewicz, P., et al., (1994) Plant Molecular
Biology 25(6): 989-94), into which a phosphinothricin resistance
under the control of the NOS promoter had been inserted before
the NOS terminator. (Figure 9, construct XIV).
Example 8:
Generation of transgenic Arabidopsis thaliana plants
Wild-type Arabidopsis thaliana plants (cv. Columbia) were
transformed with Agrobacterium tumefaciens strain (EHA105) on the
basis of a modification of Clough's and Bent's vacuum
infiltration method (Clough, S. and Bent A., Plant J.
16(6):735-43, 1998)and Bechtold, et al. (Bechtold, N., et al.,
CRAcad Sci Paris. 1144(2):204-212, 1993). The Agrobacterium
tumefaciens cells used had previously been transformed with
plasmids pZPNBN-MAAIanti or pZPNBN-FAAHanti.
Seeds of the primary transformants were screened on the basis of
their phosphinothricin resistance by planting seed by hand and
spraying the seedlings with the herbicide Basta
(phosphinothricin). Basta-resistant seedlings were singled out
and used for biochemical analysis as fully-developed plants.
Example 9: Generation of transgeniic oilseed rape (Brassica
napus) plants
The generation of transgenic oilseed rape plants followed in
principle the procedure of Bade, J.B. and Damm, B. (Bade, J.B.
and Damm, B. (1995) in: Gene Transfer to Plants, Potrykus, I. and
Spangenberg, G., eds, Springer Lab Manual, Springer Verlag, 1995,
30-38), which also indicates the composition of the media and
buffers used.
The transformation was carried out with the Agrobacterium
tumefaciens strain EHA105. Either plasmid pPTVHPPDopHGDanti
(Figure 4, construct VI) or cultures of agrobacteria with
CA 02422760 2003-03-17
38
plasmids pPTVHGDanti (Figure 4, construct V) and pPZP200HPPDop
(Figure 5, construct VTI) which were mixed after culturing were
used for the transformation. Seeds of Brassica napus var. Westar
were surface-sterilized with 70~ strength ethanol (v/v), washed
for 10 minutes with water at 55°C, incubated for 20 minutes in 1~
strength hypochlorite solution (25~ v/v Teepol, 0.1 ~ v/v Tween
20) and washed six times with sterile water for in each case 20
minutes. The seeds were dried for three days on filter paper and
IO-15 seeds were germinated in a glass flask containing 15 ml of
termination medium. Roots and apices were removed from several
seedlings (approx. size 10 cm), and the hypocotyls which remained
were cut into sections approx. 6 mm long. The approx. 600
explants thus obtained were washed fcr 30 minutes with 50 ml of
basal medium and transferred 11110 a 300 ml flask. After addition
of 100 ml of callus induction medium, the cultures were incubated
for 24 hours at 100 rpm.
Overnight cultures of the Agrobacterium strains were set up in
Luria broth supplemented with kanamycin (20 mg/1) at 29°C, and
2 ml of this were incubated in 50 ml of Luria broth medium
without kanamycin for 4 hours at 29°C until an OD6oo of 0.4 -0.5
was reached. After the culture had been pelleted for 25 minutes
at 2000 rpm, the cell pellet was resuspended in 25 ml of basal
medium. The bacterial concentration of the solution was brought
to an OD6oo of 0.3 by adding more basal medium. Far the
cotransformation, the solution of the two strains was mixed in
equal parts.
The callus induction medium was removed from the oilseed rape
explants using sterile pipettes, 50 ml of Agrobacterium solution
were added, and the reaction wass mixed carefully and incubated
for 20 minutes. The agrobacterial suspension was removed, the
oilseed rape explants were washed for 1 minute with 50 ml of
callus induction medium, and 100 ml of callus induction medium
were subsequently added. Coculturing was carried out for 24 hours
on an orbital shaker at 100 rpm. Coculturing was stopped by
removing the callus induction medium and the explants were washed
twice for in each case 1 minute with 25 ml and twice for
60 minutes with in each case 100 ml of wash medium at 100 rpm.
The wash medium together with the explants was transferred into
15 cm Petri dishes, and the medium was removed using sterile
pipettes.
For regeneration, in each case 20-30 explants were transferred
into 90 mm Petri dishes containing 25 ml of shoot induction
medium supplement with phosphinothricin. The Petri dishes were
sealed with 2 layers of Leukopor and incubated at 25°C and
081,~~00017 CA 02422760 2003-03-17
39
2000 lux at photoperiods of 16 hours light / 8 hours darkness.
Every 12 days, the calli which developed were transferred to
fresh Petri dishes containing shoot induction medium. All further
steps for the regeneration of intact plants were carried out as
described by Bade, J.B and Damm, B. (in: Gene Transfer to Plants,
Potrykus, I. and Spangenberg, G., eds, Springer Lab Manual,
Springer Verlag, 1995, 30-38).
Example 10:
Analysis of the transgenic plants
To verify that inhibition of HGD, MAAI and/or FAAH affects
vitamin E biosynthesis in the transgenic plants, the tocopherol
and tocotrienol contents in leaves and seeds of the plants
(Arabidopsp.s thaliana, Brassica napus) which had been transformed
with the above-described constructs were analyzed. To this end,
the transgenic plants are grown in the greenhouse, and plants
which express the antisense RNA of HGD, MAAI and/or FAAH are
analyzed by means of a Northern blot analysis. The tocopherol
content and the tocotrienol content in the leaves and seeds of
these plants is determined. The plant material was disrupted by
three indubations for 15 minutes in the Eppendorf shaker at 30°C,
1000 .rpm in 100 methanol, and the supernatants obtained in each
case were combined. Further incubation steps revealed no further
liberation of tocopherols or tocotrienols. To avoid oxidation,
the extracts obtained were analyzed directly after extraction
with the aid of a Waters Allience 2690 HPLC system. Tocopherols
and tocotrienols were separated using a reversed-phase column
(ProntoSil 200-3-C30, Bischoff) using a mobile phase of 100
methanol and identified with reference to standards (Merck). The
detection system used was the fluorescence of the substances
(excitation 295 nm, emission 320 nm), which was detected with the
aid of Jasco fluorescence detectors FP 920.
In all cases, the tocopherol and/or tocotrienol concentration in
transgenic plants which additionally express a nucleic acid
according to the invention is increased in comparison with
untransformed plants.
45
CA 02422760 2003-03-17
1
SEQUENCE LISTING
<110> SunGene GmbH & Co. KGaA
<120> Improved processes for vitamin E biosynthesis
<130> NAE445/2000
<140>
<141>
<160> 44
<170> PatentIn Ver. 2.1
<210> 1
<211> 2151
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> gene
<222> (1)..(2151)
<223> gene for homogentisate-1,2-dioxygenase (HGD)
<400> 1
atggaagaga agaagaagga gcttgaagag ttgaagtatc aatcaggttt tggtaaccac 60
ttctcatcgg aagcaatcgc cggagcttta ccgttagatc agaacagtcc tcttctttgt 120
ccttacggtc tttacgccga acagatctcc ggtacttctt tcacttctcc tcgcaagctc 180
aatcaaagaa ggtacatcat catttcaatt gtaagttttg gataatttcg ttgaattgat 240
tgatcttcat cttgtttttt ttttcagttg gttgtaccgg gttaaaccat cggttacaca 300
tgaaccgttc aagcctcgtg taccagctca taagaagctt gtgagtgagt ttgatgcatc 360
aaatagtcgt acgaatccga ctcagcttcg gtggagacct gaggatattc ctgattcgga 420
gattgatttc gttgatgggt tatttaccat ttgtggagct ggaagctcgt ttcttcgcca 480
tggcttcgct attcacatgt aaaaaactct tctttttatt ttggtatctt tggtgtagat 540
cagtgataca taaagtaatg atcttttgta ttcattttgt tttgaaggta tgtggctaac 600
acaggaatga aagactccgc attttgcaac gctgatggtg acttcttgtt agttcctcaa 660
acaggaagta agttagtagt cccaatgcct taccttacca catctttggg aaataaagtc 720
agtcatgtat tgagaatgga ttcaagatag tcttggatca gttctgatag tttgagtggg 780
tgttttaggg ctatggattg aaactgagtg tggaaggctt ttggtaactc ctggtgagat 840
tgctgttata ccacaaggtt tccgtttctc catagattta ccggatggga agtctcgtgg 900
ttatgttgct gaaatctatg gggctcattt tcagcttcct gatcttggac caataggtac 960
tcttgagttc ttttagattc agccggaata acatggattc tccgcaagaa tcttattggt 1020
ggatgtggac aggtgctaat ggtcttgctg catcaagaga ttttcttgca ccaacagcat 1080
ggtttgagga tggattgcgg cctgaataca caattgttca gaagtttggc ggtgaactct 1140
ttactgctaa acaagatttc tctccattca atgtggttgc ctggcatggc aattacgtgc 1200
cttataaggt gagtacattg tttattgagc ctaatcttgt aaaacgttaa tgcattgttt 1260
ttctgagaat ttcaatttct gtctgcagta tgacctgaag aagttctgtc catacaacac 1320
tgtgctttta gatcatggag atccatctat aaatacaggt tggtggtcat ctgcgctaaa_1380
tcgattcttc tttttgtttt gttatgggtt ggttacttgt tctttattgt aatcacactc 1440
tttgggtgaa ttattgtact ctcagtcctt acagcaccaa ctgataaacc tggtgtggcc 1500
~SunGene GmbH & Co. KgaA 20000445 O.Z. 0817/00017 DE
2
ttgcttgatt ttgtcatatt tcctcctcga tggttggttg ctgagcatac ttttcgacct 1560
ccttactatc atcgtaactg catgagtgaa tttatgggct taatctacgg tgcatacgag 1620
gtaagctgct tgaagttcct gcttctgcaa atcattagct ggcttgtgtt atcctcctac 1680
tgaaatctgt aaactgactc caccattcac aggcgaaagc tgatggattt ctccctggcg 1740
gtgcaagtct tcatagctgt atgacacctc atggtccaga tactaccacg tacgaggtat 1800
caatccatct tatgcacagc agcaactaca cgtttgattt cattttcctc cgagatcatg 1860
tctaaatcta acccctgaat gtaaaattaa gtctgaagca tttttataat tgttttgtag 1920
gcgacaattg ctcgagtaaa tgcaatggct ccttctaaac tcacaggtac gatggctttc 1980
atgttcgaat cagcattgat ccctagagtc tgtcattggg ctctggagtc tcctttcctg 2040
gatcacgact actaccagtg ttggattggc ctcaagtctc atttctcgcg cataagcttg 2100
gacaagacaa atgttgaatc aacagagaaa gaaccaggag cttcggagta a 2151
<210> 2
<211> 1386
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> CDS
<222> (1)..(1383)
<223> cDNA coding for homogentisate-1,2-dioxygenase
(HGD)
<400> 2
atg gaa gag aag aag aag gag ctt gaa gag ttg aag tat caa tca ggt 48
Met Glu Glu Lys Lys Lys Glu Leu Glu Glu Leu Lys Tyr Gln Ser Gly
1 5 10 15
ttt ggt aac cac ttc tca tcg gaa gca atc gcc gga get tta ccg tta 96
Phe Gly Asn His Phe Ser Ser Glu Ala Ile Ala Gly Ala Leu Pro Leu
20 25 30
gat cag aac agt cct ctt ctt tgt cct tac ggt ctt tac gcc gaa cag 144
Asp Gln Asn Ser Pro Leu Leu Cys Pro Tyr Gly Leu Tyr Ala Glu Gln
35 40 45
atc tcc ggt act tct ttc act tct cct cgc aag ctc aat caa aga agt 192
Ile Ser Gly Thr Ser Phe Thr Ser Pro Arg Lys Leu Asn Gln Arg Ser
50 55 60
tgg ttg tac cgg gtt aaa cca tcg gtt aca cat gaa ccg ttc aag cct 240
Trp Leu Tyr Arg Val Lys Pro Ser Val Thr His Glu Pro Phe Lys Pro
65 70 75 80
cgt gta cca get cat aag aag ctt gtg agt gag ttt gat gca tca aat 288
Arg Val Pro Ala His Lys Lys Leu Val Ser Glu Phe Asp Ala Ser Asn
85 90 95
agt cgt acg aat ccg act cag ctt cgg tgg aga cct gag gat att cct 336
Ser Arg Thr Asn Pro Thr Gln Leu Arg Trp Arg Pro Glu Asp Ile Pro
100 105 110
CA 02422760 2003-03-17
'SunGene GmbH & Co. KgaA 20000445 0.2. 0817/00017 DE
3
gat tcg gag att gat ttc gtt gat ggg tta ttt acc att tgt gga get 384
Asp Ser Glu Ile Asp Phe Val Asp Gly Leu Phe Thr Ile Cys Gly Ala
115 120 125
gga agc tcg ttt ctt cgc cat ggc ttc get att cac atg tat gtg get 432
Gly Ser Ser Phe Leu Arg His Gly Phe Ala Ile His Met Tyr Val Ala
130 135 140
aac aca gga atg aaa gac tcc gca ttt tgc aac get gat ggt gac ttc 480
Asn Thr Gly Met Lys Asp Ser Ala Phe Cys Asn Ala Asp Gly Asp Phe
145 150 155 160
ttg tta gtt cct caa aca gga agg cta tgg att gaa act gag tgt gga 528
Leu Leu Val Pro Gln Thr Gly Arg Leu Trp Ile Glu Thr Glu Cys Gly
165 170 175
agg ctt ttg gta act cct ggt gag att get gtt ata cca caa ggt ttc 576
Arg Leu Leu Val Thr Pro Gly Glu Ile Ala Val Ile Pro Gln Gly Phe
180 185 190
cgt ttc tcc ata gat tta ccg gat ggg aag tct cgt ggt tat gtt get 624
Arg Phe Ser Ile Asp Leu Pro Asp Gly Lys Ser Arg Gly Tyr Val Ala
195 200 205
gaa atc tat ggg get cat ttt cag ctt cct gat ctt gga cca ata ggt 672
Glu Ile Tyr Gly Ala His Phe Gln Leu Pro Asp Leu Gly Pro Ile Gly
210 215 220
get aat ggt ctt get gca tca aga gat ttt ctt gca cca aca gca tgg 720
Ala Asn Gly Leu Ala Ala Ser Arg Asp Phe Leu Ala Pro Thr Ala Trp
225 230 235 240
ttt gag gat gga ttg cgg cct gaa tac aca att gtt cag aag ttt ggc 768
Phe Glu Asp Gly Leu Arg Pro Glu Tyr Thr Ile Va1 Gln Lys Phe Gly
245 250 255
ggt gaa ctc ttt act get aaa caa gat ttc tct cca ttc aat gtg gtt 816
Gly Glu Leu Phe Thr Ala Lys Gln Asp Phe Ser Pro Phe Asn Val Val
260 265 270
gcc tgg cat ggc aat tac gtg cct tat aag tat gac ctg aag aag ttc 864
Ala Trp His Gly Asn Tyr Val Pro Tyr Lys Tyr Asp Leu Lys Lys Phe
275 280 285
tgt cca tac aac act gtg ctt tta gat cat gga gat cca tct ata aat 912
Cys Pro Tyr Asn Thr Val Leu Leu Asp His Gly Asp Pro Ser Ile Asn
290 295 300
aca gtc ctt aca gca cca act gat aaa cct ggt gtg gcc ttg ctt gat 960
Thr Val Leu Thr Ala Pro Thr Asp Lys Pro Gly Val Ala Leu Leu Asp
305 310 315 320
CA 02422760 2003-03-17
'SunGene GmbH & Co. KgaA 20000445 O.Z. 0817/00017 DE
4
ttt gtc ata ttt cct cct cga tgg ttg gtt get gag cat act ttt cga 1008
Phe Val Ile Phe Pro Pro Arg Trp Leu Val Ala Glu His Thr Phe Arg
325 330 335
cct cct tac tat cat cgt aac tgc atg agt gaa ttt atg ggc tta atc 1056
Pro Pro Tyr Tyr His Arg Asn Cys Met Ser Glu Phe Met Gly Leu Ile
340 345 350
tac ggt gca tac gag gcg aaa get gat gga ttt ctc cct ggc ggt gca 1104
Tyr Gly Ala Tyr Glu Ala Lys Ala Asp Gly Phe Leu Pro Gly Gly Ala
355 360 365
agt ctt cat agc tgt atg aca cct cat ggt cca gat act acc acg tac 1152
Ser Leu His Ser Cys Met Thr Pro His Gly Pro Asp Thr Thr Thr Tyr
370 375 380
gag gcg aca att get cga gta aat gca atg get cct tct aaa ctc aca 1200
Glu Ala Thr Ile Ala Arg Val Asn Ala Met Ala Pro Ser Lys Leu Thr
385 390 395 400
ggt acg atg get ttc atg ttc gaa tca gca ttg atc cct aga gtc tgt 1248
Gly Thr Met Ala Phe Met Phe Glu Ser Ala Leu Ile Pro Arg Val Cys
405 410 415
cat tgg get ctg gag tct cct ttc ctg gat cac gac tac tac cag tgt 1296
His Trp Ala Leu Glu Ser Pro Phe Leu Asp His Asp Tyr Tyr Gln Cys
420 425 430
tgg att ggc ctc aag tct cat ttc tcg cgc ata agc ttg gac aag aca 1344
Trp Ile Gly Leu Lys Ser His Phe Ser Arg Ile Ser Leu Asp Lys Thr
435 440 445
aat gtt gaa tca aca gag aaa gaa cca gga get tcg gag taa 1386
Asn Val Glu Ser Thr Glu Lys Glu Pro Gly Ala Ser Glu
450 455 460
<210> 3
<211> 461
<212> PRT
<213> Arabidopsis thaliana
<400> 3
Met Glu Glu Lys Lys Lys Glu Leu Glu Glu Leu Lys Tyr Gln Ser Gly
1 5 10 15
Phe Gly Asn His Phe Ser Ser Glu Ala Ile Ala Gly Ala Leu Pro Leu
20 25 30
Asp Gln Asn Ser Pro Leu Leu Cys Pro Tyr Gly Leu Tyr Ala Glu Gln
35 40 45
CA 02422760 2003-03-17
'SunGene GmbH & Co. Kga.A 20000445 O.Z. 0817/00017 DE
Ile Ser Gly Thr Ser Phe Thr Ser Pro Arg Lys Leu Asn Gln Arg Ser
50 55 60
Trp Leu Tyr Arg Val Lys Pro Ser Val Thr His Glu Pro Phe Lys Pro
65 70 75 80
Arg Val Pro Ala His Lys Lys Leu Val Sex Glu Phe Asp Ala Ser Asn
85 90 95
Ser Arg Thr Asn Pro Thr Gln Leu Arg Trp Arg Pro Glu Asp IIe Pro
100 105 110
Asp Ser Glu Ile Asp Phe Val Asp Gly Leu Phe Thr Ile Cys Gly Ala
115 120 125
Gly Ser Ser Phe Leu Arg His Gly Phe Ala Ile His Met Tyr Val Ala
I30 135 240
Asn Thr Gly Met Lys Asp Ser Ala Phe Cys Asn Ala Asp Gly Asp Phe
145 150 155 160
Leu Leu Val Pro Gln Thr Gly Arg Leu Trp Ile Glu Thr Glu Cys Gly
165 170 175
Arg Leu Leu Val Thr Pro Gly Glu Ile Ala Val Ile Pro Gln Gly Phe
180 185 190
Arg Phe Ser Ile Asp Leu Pro Asp Gly Lys Ser Arg Gly Tyr Val Ala
195 200 205
Glu Ile Tyr Gly Ala His Phe Gln Leu Pro Asp Leu Gly Pro Ile Gly
210 215 220
Ala Asn Gly Leu Ala Ala Ser Arg Asp Phe Leu Ala Pro Thr Ala Trp
225 230 235 240
Phe Glu Asp Gly Leu Arg Pro Glu Tyr Thr Ile Val Gln Lys Phe Gly
245 250 255
Gly Glu Leu Phe Thr Ala Lys Gln Asp Phe Ser Pro Phe Asn Val Val
260 265 270
Ala Trp His Gly Asn Tyr Val Pro Tyr Lys Tyr Asp Leu Lys Lys Phe
27S 280 285
Cys Pro Tyr Asn Thr Val Leu Leu Asp His Gly Asp Pro Ser Ile Asn
290 295 300
Thr Val Leu Thr AIa Pro Thr Asp Lys Pro GIy Val Ala Leu Leu Asp
305 310 315 320
CA 02422760 2003-03-17
w SunGene GmbH & Co. Kga.A 20000445 O.Z. 0817/00017 DE
6
Phe Val Ile Phe Pro Pro Arg Trp Leu Val Ala Glu His Thr Phe Arg
325 330 335
Pro Pro Tyr Tyr His Arg Asn Cys Met Ser Glu Phe Met Gly Leu Ile
340 345 350
Tyr Gly Ala Tyr Glu Ala Lys Ala Asp Gly Phe Leu Pro Gly Gly Ala
355 360 365
Ser Leu His Ser Cys Met Thr Pro His Gly Pro Asp Thr Thr Thr Tyr
370 375 380
Glu Ala Thr Ile Ala Arg Val Asn Ala Met Ala Pro Ser Lys Leu Thr
385 390 395 400
Gly Thr Met Ala Phe Met Phe Glu Ser Ala Leu Ile Pro Arg Val Cys
405 410 415
His Trp Ala Leu Glu Ser Pro Phe Leu Asp His Asp Tyr Tyr Gln Cys
420 425 430
Trp Ile Gly Leu Lys Ser His Phe Ser Arg Ile Ser Leu Asp Lys Thr
435 440 445
Asn Val Glu Ser Thr Glu Lys Glu Pro Gly Ala Ser Glu
450 455 460
<210> 4
<211> 1227
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> CDS
<222> (1)..(1224)
<223> cDNA coding for fumarylacetoacetate hydrolase
(FAAH)
<400> 4
atg gcg ttg ctg aag tct ttc atc gat gtt ggc tca gac tcg cac ttc 48
Met Ala Leu Leu Lys Ser Phe Ile Asp Val Gly Ser Asp Ser His Phe
1 5 10 15
cct atc cag aat ctc cct tat ggt gtc ttc aaa ccg gaa tcg aac tca 96
Pro Ile Gln Asn Leu Pro Tyr Gly Val Phe Lys Pro Glu Ser Asn Ser
20 25 30
act cct cgt cct gcc gtc get atc ggc gat ttg gtt ctg gac ctc tcc 144
Thr Pro Arg Pro Ala Val Ala Ile Gly Asp Leu Val Leu Asp Leu Ser
35 40 45
CA 02422760 2003-03-17
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7
get atc tct gaa get ggg ctt ttc gat ggt ctg atc ctt aag gac gca 192
Ala Ile Ser Glu Ala Gly Leu Phe Asp Gly Leu Ile Leu Lys Asp Ala
50 55 60
gat tgc ttt ctt cag cct aat ttg aat aag ttc ttg gcc atg gga cgg 240
Asp Cys Phe Leu Gln Pro Asn Leu Asn Lys Phe Leu Ala Met Gly Arg
65 70 75 80
cct gcg tgg aag gaa gcg cgt tct acg ctg caa aga atc ttg tca ttt 288
Pro Ala Trp Lys Glu Ala Arg Ser Thr Leu Gln Arg Ile Leu Ser Phe
85 90 95
ttg tta ttt ggc ttc aag gtt ttg gtt ttg gta tgt ttt cat gca get 336
Leu Leu Phe Gly Phe Lys Val Leu Val Leu Val Cys Phe His Ala Ala
100 105 110
aat gaa cct atc ttg cga gac aat gat gtt ttg agg aga aaa tca ttc 384
Asn Glu Pro Ile Leu Arg Asp Asn Asp Val Leu Arg Arg Lys Ser Phe
115 120 125
cat cag atg agt aaa gtg gaa atg att gtt cct atg gtg att ggg gac 432
His Gln Met Ser Lys Val Glu Met Ile Val Pro Met Val Ile Gly Asp
130 135 140
tat aca gac ttc ttt gca tct atg cat cac gcg aag aac tgc gga ctt 480
Tyr Thr Asp Phe Phe Ala Ser Met His His Ala Lys Asn Cys Gly Leu
145 150 155 160
atg ttc cgt ggg cct gag aat gcg ata aac cca aat tgg ttt cgt ctt 528
Met Phe Arg Gly Pro Glu Asn Ala Ile Asn Pro Asn Trp Phe Arg Leu
165 170 175
ccc att gca tat cat gga cgg gca tca tct att gtc atc tct ggg act 576
Pro Ile Ala Tyr His Gly Arg Ala Ser Ser Ile Val Ile Ser Gly Thr
180 185 190
gac att att cga cca aga ggt cag ggc cat cca caa gga aac tct gaa 624
Asp Ile Ile Arg Pro Arg Gly Gln Gly His Pro Gln Gly Asn Ser Glu
195 200 205
cca tat ttt gga cct tcg aag aaa ctt gat ttt gag ctt gag atg get 672
Pro Tyr Phe Gly Pro Ser Lys Lys Leu Asp Phe Glu Leu Glu Met Ala
210 215 220
get gtg gtt ggt cca gga aat gaa ttg gga aag cct att gac gtg aat 720
Ala Val Val Gly Pro Gly Asn Glu Leu Gly Lys Pro Ile Asp Val Asn
225 230 235 240
aat gca gcc gat cat ata ttt ggt cta tta ctg atg aat gac tgg agt 768
Asn Ala Ala Asp His Ile Phe Gly Leu Leu Leu Met Asn Asp Trp Ser
245 250 255
CA 02422760 2003-03-17
~SunGene GmbH & Co. KgaA 20000445 O.Z. 0817/00017 DE
8
get agg gat att cag gcg tgg gag tat gta cct ctt ggt cct ttc ctg 816
Ala Arg Asp Ile Gln Ala Trp Glu Tyr Val Pro Leu Gly Pro Phe Leu
260 265 270
ggg aag agt ttt ggg act act ata tcc cct tgg att gtt acc ttg gat 864
Gly Lys Ser Phe Gly Thr Thr Ile Ser Pro Trp Ile Val Thr Leu Asp
275 280 285
gcg ctt gag cct ttt ggt tgt caa get ccc aag cag gat cca cct cca 912
Ala Leu Glu Pro Phe Gly Cys Gln Ala Pro Lys Gln Asp Pro Pro Pro
290 295 300
ttg cca tat ttg get gag aaa gag tct gta aat tac gat atc tcc ttg 960
Leu Pro Tyr Leu Ala Glu Lys Glu Ser Val Asn Tyr Asp Ile Ser Leu
305 310 315 320
gag cta gca cac cat acc gtt aac ggt tgc aat ttg agg cct ggt gat 1008
Glu Leu Ala His His Thr Val Asn Gly Cys Asn Leu Arg Pro Gly Asp
325 330 335
ctc ctt ggc aca gga acc ata agc gga ccg gag cca gat tca tat ggg 1056
Leu Leu Gly Thr Gly Thr Ile Ser Gly Pro Glu Pro Asp Ser Tyr Gly
340 345 350
tgc cta ctt gag ttg aca tgg aat gga cag aaa cct cta tca ctc aat 1104
Cys Leu Leu Glu Leu Thr Trp Asn Gly Gln Lys Pro Leu Ser Leu Asn
355 360 365
gga aca act cag acg ttt ctc gaa gac gga gac caa gtc acc ttc tca 1152
Gly Thr Thr Gln Thr Phe Leu Glu Asp GIy Asp Gln Val Thr Phe Ser
370 375 380
ggt gta tgc aag gga gat ggt tac aat gtt ggg ttt gga aca tgc aca 1200
Gly Val Cys Lys Gly Asp Gly Tyr Asn Val Gly Phe Gly Thr Cys Thr
385 390 395 400
ggg aaa att gtt cct tca ccg cct tga 1227
Gly Lys Ile Val Pro Ser Pro Pro
405
<210> 5
<211> 408
<212> PRT
<213> Arabidopsis thaliana
<400> 5
Met Ala Leu Leu Lys Ser Phe Ile Asp Val Gly Ser Asp Ser His Phe
1 5 10 15
Pro Ile Gln Asn Leu Pro Tyr Gly Val Phe Lys Pro Glu Ser Asn Ser
20 25 30
CA 02422760 2003-03-17
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Thr Pro Arg Pro Ala Val Ala Ile Gly Asp Leu Val Leu Asp Leu Ser
35 40 45
Ala Ile Ser Glu Ala Gly Leu Phe Asp Gly Leu Ile Leu Lys Asp Ala
50 55 60
Asp Cys Phe Leu Gln Pro Asn Leu Asn Lys Phe Leu Ala Met Gly Arg
65 70 75 80
Pro Ala Trp Lys Glu Ala Arg Ser Thr Leu Gln Arg IIe Leu Ser Phe
85 90 95
Leu Leu Phe Gly Phe Lys Val Leu Val Leu Val Cys Phe His Ala Ala
100 105 110
Asn Glu Pro Ile Leu Arg Asp Asn Asp Val Leu Arg Arg Lys Ser Phe
115 120 225
His Gln Met Ser Lys Val Glu Met Ile Val Pro Met Val Ile Gly Asp
130 135 140
Tyr Thr Asp Phe Phe Ala Ser Met His His Ala Lys Asn Cys Gly Leu
145 150 155 160
Met Phe Arg Gly Pro Glu Asn Ala Ile Asn Pro Asn Trp Phe Arg Leu
165 170 175
Pro Ile Ala Tyr His Gly Arg Ala Ser Ser Ile Val Ile Ser Gly Thr
180 185 190
Asp Ile Ile Arg Pro Arg Gly Gln Gly His Pro Gln Gly Asn Ser Glu
195 200 205
Pro Tyr Phe Gly Pro Ser Lys Lys Leu Asp Phe Glu Leu Glu Met Ala
210 215 220
Ala Val Val Gly Pro Gly Asn Glu Leu Gly Lys Pro Ile Asp Val Asn
225 230 235 240
Asn Ala Ala Asp His Ile Phe Gly Leu Leu Leu Met Asn Asp Trp Ser
245 250 255
Ala Arg Asp Ile Gln Ala Trp Glu Tyr Val Pro Leu Gly Pro Phe Leu
260 265 270
Gly Lys Ser Phe Gly Thr Thr Ile Ser Pro Trp Ile Val Thr Leu Asp
275 280 285
Ala Leu Glu Pro Phe Gly Cys Gln Ala Pro Lys Gln Asp Pro Pro Pro
290 295 300
CA 02422760 2003-03-17
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Leu Pro Tyr Leu Ala Glu Lys Glu Ser Val Asn Tyr Asp Ile Ser Leu
305 310 315 320
Glu Leu Ala His His Thr Val Asn Gly Cys Asn Leu Arg Pro Gly Asp
325 330 335
Leu Leu Gly Thr Gly Thr Ile Ser Gly Pro Glu Pro Asp Ser Tyr Gly
340 345 350
Cys Leu Leu Glu Leu Thr Trp Asn Gly Gln Lys Pro Leu Ser Leu Asn
355 360 365
Gly Thr Thr Gln Thr Phe Leu Glu Asp Gly Asp Gln Val Thr Phe Ser
370 375 380
Gly Val Cys Lys Gly Asp Gly Tyr Asn Val Gly Phe Gly Thr Cys Thr
385 390 395 400
Gly Lys Ile Val Pro Ser Pro Pro
405
<210> 6
<211> 1721
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> gene
<222> (9)..(1713)
<223> gene for maleylacetoacetate isomerase (MA.AI)
<220>
<221> misc feature
<222> (.1-)..($)
<223> restriction site linker
<220>
<221> misc feature
<222> (1714)..(1721)
<223> restriction site linker
<400> 6
atgtcgacat gtcttatgtt accgattttt atcaggcgaa gttgaagctc tactcttact 60
ggagaagctc atgtgctcat cgcgtccgta tcgccctcac tttaaaaggt accagccaat 120
gattttattc ttttcttgtg agcaattctt tgatctgaat ttggttcttg ttcgattttc 180
attagggctt gattatgaat atataccggt taatttgctc aaaggggatc aatccgattc 240
aggtgcgtag tttctaggtt atattgaact ttatttgaag taacattgta aagataagaa 300
tggtaagtaa ctgagatttc ttatgttaga cttagaagtt tattcgtttt ggttctctag 360
atttcaagaa gatcaatcca atgggcactg taccagcgct tgttgatggt gatgttgtga 420
ttaatgactc tttcgcaata ataatggtca gtagtaacac atccatttag tttgtttggt 480
tttgttgatg aaaaggaaca ttcgtttatt cgtcttgttg tttttcaaat ggacagtacc 540
CA 02422760 2003-03-17
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11
tggatgataa gtatccggag ccaccgctgt taccaagtga ctaccataaa cgggcggtaa 600
attaccaggt atcttcgatc ctttgtcttc agatgatgat gtgttgccat catctgcaaa 660
accatgtagt taagtccaaa tgtagtgaac attatcagct ttagattgcg agtgtgatcg 720
ttgttcttat tttgtatatt tcaggcgacg agtattgtca tgtctggtat acagcctcat 780
caaaatatgg ctctttttgt gagaagatga gattaatgta atggattcta ctaatggagg 840
ttctataaca aagcaaacat agttacattt tgtcattttt tttaacagag gtatctcgag 900
gacaagataa atgctgagga gaaaactgct tggattacta atgctatcac aaaaggattc 960
acaggtatga tatctctaat ctacctatac gtaatcaaga accaagacat atgttcaaaa 1020
tgtgattttg ttgatattgt ggttgtacag gtttataacg acctgtctga taatgtctca 1080
tatgtccttc agctctcgag aaactgttgg tgagttgcgc tggaaaatac gcgactggtg 1140
atgaagttta cttggtatgt ctctaaatct ccctggataa tctctatggt actactctct 1200
tctttattac aatgaagcat tgttttgcag gctgatcttt tcctagcacc acagatccac 1260
gcagcattca acagattcca tattaacatg gtacttttcc tcagctaatc tcttctcctg 1320
gtacctagat attgcattgt atatcccccc aaattccatg gaatccttga tcagagtttt 1380
aaggtagcat gaaccaaatg ttatctctgt ctcacacttt cacattcaca gagtaacata 1440
gacgtaatac tcagtttcat aacttttttt cctcgcatca cttggttttc atctctacaa 1500
ttttgttgta taggaaccat tcccgactct tgcaaggttt tacgagtcat acaacgaact 1560
gcctgcattt caaaatgcag tcccggagaa gcaaccagat actccttcca ccatctgatt 1620
ctgtgaaccg taagcttctc tcagtctcag ctcaataaaa tctcttagga aacaacaaca 1680
acaccttgaa cttaaatgta tcatatgaac cagggatcca t 1721
<210> 7
<211> 1238
<212> DNA
<213> Escherichia coli
<220>
<221> gene
<222> (7)..(1232)
<223> tyrA gene coding for bifunctional chorismate
mutase / prephenate dehydrogenase
<220>
<221> CDS
<222> (25)..(1143)
<220>
<221> misc feature
<222> (1)..(6)
<223> restriction site linker
<220>
<221> misc feature
<222> (1233)..(1238)
<223> restriction site linker
<400> 7
cccgggtggc ttaagaggtt tatt atg gtt get gaa ttg acc gca tta cgc 51
Met Val Ala Glu Leu Thr Ala Leu Arg
1 5
CA 02422760 2003-03-17
~SunGene GmbH & Co. Kga.A 20000445 O.Z. 0$17/00017 DE
12
gat caa att gat gaa gtc gat aaa gcg ctg ctg aat tta tta gcg aag 99
Asp Gln Ile Asp Glu Val Asp Lys Ala Leu Leu Asn Leu Leu Ala Lys
15 20 25
cgt ctg gaa ctg gtt get gaa gtg ggc gag gtg aaa agc cgc ttt gga 147
Arg Leu Glu Leu Val Ala Glu Val Gly Glu Val Lys Ser Arg Phe Gly
30 35 40
ctg cct att tat gtt ccg gag cgc gag gca tct atg ttg gcc tcg cgt 195
Leu Pro Ile Tyr Val Pro Glu Arg Glu Ala Ser Met Leu Ala Ser Arg
45 50 55
cgt gca gag gcg gaa get ctg ggt gta ccg cca gat ctg att gag gat 243
Arg Ala Glu Ala Glu Ala Leu Gly Val Pro Pro Asp Leu Ile Glu Asp
60 65 70
gtt ttg cgt cgg gtg atg cgt gaa tct tac tcc agt gaa aac gac aaa 291
Val Leu Arg Arg Val Met Arg Glu Ser Tyr Ser Ser Glu Asn Asp Lys
75 80 85
gga ttt aaa aca ctt tgt ccg tca ctg cgt ccg gtg gtt atc gtc ggc 339
Gly Phe Lys Thr Leu Cys Pro Ser Leu Arg Pro Val Val Ile Val Gly
90 95 100 105
ggt ggc ggt cag atg gga cgc ctg ttc gag aag atg ctg acc ctc tcg 387
Gly Gly Gly Gln Met Gly Arg Leu Phe Glu Lys Met Leu Thr Leu Ser
110 115 120
ggt tat cag gtg cgg att ctg gag caa cat gac tgg gat cga gcg get 435
Gly Tyr Gln Val Arg Ile Leu Glu Gln His Asp Trp Asp Arg Ala Ala
125 130 135
gat att gtt gcc gat gcc gga atg gtg att gtt agt gtg cca atc cac 483
Asp Ile Val Ala Asp Ala Gly Met Val Ile Val Ser Val Pro Ile His
140 145 150
gtt act gag caa gtt att ggc aaa tta ccg cct tta ccg aaa gat tgt 531
Val Thr Glu Gln Val Ile Gly Lys Leu Pro Pro Leu Pro Lys Asp Cys
155 160 165
att ctg gtc gat ctg gca tca gtg aaa aat ggg cca tta cag gcc atg 579
Ile Leu Val Asp Leu Ala Ser Val Lys Asn Gly Pro Leu Gln Ala Met
170 175 280 185
ctg gtg gcg cat gat ggt ccg gtg ctg ggg cta cac ccg atg ttc ggt 627
Leu Val Ala His Asp Gly Pro Val Leu Gly Leu His Pro Met Phe Gly
190 195 200
ccg gac agc ggt agc ctg gca aag caa gtt gtg gtc tgg tgt gat gga 675
Pro Asp Ser Gly Ser Leu Ala Lys Gln Val Val Val Trp Cys Asp Gly
205 210 215
CA 02422760 2003-03-17
.w SunGene GmbH & Co. Kga.A 20000445 O.Z. 0817/00017 DE
13
cgt aaa ccg gaa gca tac caa tgg ttt ctg gag caa att cag gtc tgg 723
Arg Lys Pro Glu Ala Tyr Gln Trp Phe Leu Glu Gln Ile Gln Val Trp
220 225 230
ggc get cgg ctg cat cgt att agc gcc gtc gag cac gat cag aat atg 771
Gly Ala Arg Leu His Arg Ile Ser Ala Val Glu His Asp Gln Asn Met
235 240 245
gcg ttt att cag gca ctg cgc cac ttt get act ttt get tac ggg ctg 819
Ala Phe Ile Gln Ala Leu Arg His Phe Ala Thr Phe Ala Tyr Gly Leu
250 255 260 265
cac ctg gca gaa gaa aat gtt cag ctt gag caa ctt ctg gcg ctc tct 867
His Leu Ala Glu Glu Asn Val Gln Leu Glu Gln Leu Leu Ala Leu Ser
270 275 280
tcg ccg att tac cgc ctt gag ctg gcg atg gtc ggg cga ctg ttt get 915
Ser Pro Ile Tyr Arg Leu Glu Leu Ala Met Val Gly Arg Leu Phe Ala
285 290 295
cag gat ccg cag ctt tat gcc gac atc att atg tcg tca gag cgt aat 963
Gln Asp Pro Gln Leu Tyr Ala Asp Ile Ile Met Ser Ser Glu Arg Asn
300 305 310
ctg gcg tta atc aaa cgt tac tat aag cgt ttc ggc gag gcg att gag 1011
Leu Ala Leu Ile Lys Arg Tyr Tyr Lys Arg Phe Gly Glu Ala Ile Glu
315 320 325
ttg ctg gag cag ggc gat aag cag gcg ttt att gac agt ttc cgc aag 1059
Leu Leu Glu Gln Gly Asp Lys Gln Ala Phe Ile Asp Ser Phe Arg Lys
330 335 340 345
gtg gag cac tgg ttc ggc gat tac gca cag cgt ttt cag agt gaa agc 1107
Val Glu His Trp Phe Gly Asp Tyr Ala Gln Arg Phe Gln Ser Glu Ser
350 355 360
cgc gtg tta ttg cgt cag gcg aat gac aat cgc cag taataatcca 1153
Arg Val Leu Leu Arg Gln Ala Asn Asp Asn Arg Gln
365 370
gtgccggatg attcacatca tccggcacct tttcatcagg ttggatcaac aggcactacg 1213
ttctcacttg ggtaacagcg tcgac 1238
<210> 8
<211> 373
<212> PRT
<213> Escherichia coli
CA 02422760 2003-03-17
~SunGene GmbH & Co. Kga.A 20000445 O.Z. 0817/00017 DE
14
<400> 8
Met Val Ala Glu Leu Thr Ala Leu Arg Asp Gln Ile Asp Glu Val Asp
10 15
Lys Ala Leu Leu Asn Leu Leu Ala Lys Arg Leu Glu Leu Val Ala Glu
20 25 30
Val Gly Glu Val Lys Ser Arg Phe Gly Leu Pro Ile Tyr Val Pro Glu
35 40 45
Arg Glu Ala Ser Met Leu Ala Ser Arg Arg Ala Glu Ala Glu Ala Leu
50 55 60
Gly Val Pro Pro Asp Leu Ile Glu Asp Val Leu Arg Arg Val Met Arg
65 70 75 80
Glu Ser Tyr Ser Ser Glu Asn Asp Lys Gly Phe Lys Thr Leu Cys Pro
85 90 95
Ser Leu Arg Pro Val Val Ile Val Gly Gly Gly Gly Gln Met Gly Arg
100 105 110
Leu Phe Glu Lys Met Leu Thr Leu Ser Gly Tyr Gln Val Arg Ile Leu
115 120 125
Glu Gln His Asp Trp Asp Arg Ala Ala Asp Ile Val Ala Asp Ala Gly
130 135 140
Met Val Ile Val Ser Val Pro Ile His Val Thr Glu Gln Val Ile Gly
145 150 155 160
Lys Leu Pro Pro Leu Pro Lys Asp Cys Ile Leu Val Asp Leu Ala Ser
165 170 175
Val Lys Asn Gly Pro Leu Gln Ala Met Leu Val Ala His Asp Gly Pro
180 185 190
Val Leu Gly Leu His Pro Met Phe Gly Pro Asp Ser Gly Ser Leu Ala
195 200 205
Lys Gln Val Val Val Trp Cys Asp Gly Arg Lys Pro Glu Ala Tyr Gln
210 215 220
Trp Phe Leu Glu Gln Ile Gln Val Trp Gly Ala Arg Leu His Arg Ile
225 230 235 240
Ser Ala Val Glu His Asp Gln Asn Met Ala Phe Ile Gln Ala Leu Arg
245 250 255
His Phe Ala Thr Phe Ala Tyr Gly Leu His Leu Ala Glu Glu Asn Val
260 265 270
CA 02422760 2003-03-17
'~'SunGene GmbH & Co. Kga.A 2000045 O.Z. 0817/00017 DE
Gln Leu Glu Gln Leu Leu Ala Leu Ser Ser Pro Ile Tyr Arg Leu Glu
275 280 285
Leu Ala Met Val Gly Arg Leu Phe Ala Gln Asp Pro Gln Leu Tyr Ala
290 295 300
Asp Ile Ile Met Ser Ser Glu Arg Asn Leu Ala Leu Ile Lys Arg Tyr
305 310 315 320
Tyr Lys Arg Phe Gly Glu Ala Ile Glu Leu Leu Glu Gln Gly Asp Lys
325 330 335
Gln Ala Phe Ile Asp Ser Phe Arg Lys VaI Glu His Trp Phe Gly Asp
340 345 350
Tyr Ala Gln Arg Phe Gln Ser Glu Ser Arg Val Leu Leu Arg Gln Ala
355 360 365
Asn Asp Asn Arg Gln
370
<210> 9
<211> 2953
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> gene
<222> (1)..(2953)
<223> gene for fumarylacetoacetate hydrolase (FAAH)
CA 02422760 2003-03-17
~SunGene GmbH & Co. Kga.A 20000445 O.Z. 0817/00017 DE
16
<400> 9
atggcgttgc tgaagtcttt catcgatgtt ggctcagact cgcacttccc tatccagaat 60
ctcccttatg gtgtcttcaa accggaatcg aactcaactc ctcgtcctgc cgtcgctatc 120
ggcgatttgg ttctggacct ctccgctatc tctgaagctg ggcttttcga tggtctgatc 180
cttaaggacg cagattgctt tcttcaggtt cgtttttccg attcctataa actcggatta 240
ctatgtagta gtaccctggg aatgtttccg taaatgattt cgaatttgct atttgaacct 300
gatctctgaa gtttgctcca tggtttattg gatagatcaa tcccgtttag ctcgaaaaaa 360
atccattgtt ctactcaatt gctcgttgct tcgattcatt atctgttaca gtttgagttt 420
tctgttcacg attttgaact tttgcaacta tgattattgc tttatgatct gacggtatag 480
tgtattgctt acacttagtg atgaggaaaa tgaggttgtg tttattttct ggtgtgtttc 540
ttttgatgtt aatattgttt agtttctgtg ctctgtttgc agcctaattt gaataagttc 600
ttggccatgg gacggcctgc gtggaaggaa gcgcgttcta cgctgcaaag aatcttgtca 660
tgtatgctct gtttgatcct attgatttat ttggattttt atggagtttt gttatttggc 720
ttcaaggttt tggttttggt atgttttcat gcagctaatg aacctatctt gcgagacaat 780
gatgttttga ggagaaaatc attccatcag atggttagta gtgtgaaatt gttttttgct 840
taaactaggg aaattgtttg tatatctgtt acttacgttt attgctgttt gatgcaaatt 900
tgcagagtaa agtggaaatg attgttccta tggtgattgg ggactataca gacttctttg 960
catctatgca tcacgcgaag aactgcggac ttatgttccg tgggcctgag aatgcgataa 1020
acccaaattg gtgcgtttat gttacttttg agctgagagt ttcttcatga aatggtcaag 1080
tcgaaaggat gactctgtat taacatgaca ttaccatatt tttcaggttt cgtcttccca 1140
ttgcatatca tggacgggca tcatctattg tcatctctgg gactgacatt attcgaccaa 1200
ggttaggaaa ttgtgtatta ttatctggtt tttggtgggc tgagaatggt tgttaagaat 1260
aattcacatg tcatatttga agtcatgcat catgcaaggt tttatgcttt gacaagaaat 1320
atagtttttt ataagatatt attacattga aaccaatatt ggcggatggt aaaatttcat 1380
gcagacaaat taataatgaa atgctaattc cagttttatc tttgcttgtt ttgctttctt 1440
ccagaggtca gggccatcca caaggaaact ctgaaccata ttttggacct tcgaagaaac 1500
ttgattttga gcttgagatg gtaagcatct gatgcctcag ttatgtggat ttgttttaca 1560
atgattcggt tgatgctttt tggtgctagt taagaataac ggcattgaca aacctctctt 1620
ttatcacatg atattcaggc tgctgtggtt ggtccaggaa atgaattggg aaagcctatt 1680
gacgtgaata atgcagccga tcatatattt ggtctattac tgatgaatga ctggagtggt 1740
actcacttaa ctatagtttt cgttgagtca tctttaacct gaccgggcat gaccggtttt 1800
tttaaatgtt tgttgttata gctagggata ttcaggcgtg ggagtatgta cctcttggtc 1860
ctttcctggg gaagagtttt ggtgagatat ttggcttcaa tactttgatt tcatttcctc 1920
tagttgaagt atatgggcaa agaacttcgg tgaatgttgt cttgttgtgt tgtagggact 1980
actatatccc cttggattgt taccttggat gcgcttgagc cttttggttg tcaagctccc 2040
aagcaggttg gtacttaggc atcacattct ttttgtgtca cgcaatcact gattctctca 2100
tgatctaact tgttcttggg gcaggatcca cctccattgc catatttggc tgagaaagag 2160
tctgtaaatt acgatatctc cttggaggta gcattcgata ttggagtttc actttttggc 2220
tttttgctat caactataac agcttatggt ggactgaact gaaataaaca tcatgttttt 2280
acctcttata ggttcaactt aaaccttctg gcagagatga ttcttgtgta ataacaaaga 2340
gcaacttcca aaacttgtga gttcctctat aatctcctac ccaattcctc catataatta 2400
aacagtttgg ttcaaactct tttaaactta ttgtgacaga tattggacca taacgcagca 2460
gctagcacac cataccgtta acggttgcaa tttgaggcct ggtgatctcc ttggcacagg 2520
aaccataagc ggaccggtaa actcttttcg aaccagttct ctcgtctact atatcacgtg 2580
atgactacac aataactcgc aaaatctttg tttcttggtt ctaaacgcag gagccagatt 2640
catatgggtg cctacttgag ttgacatgga atggacagaa acctctatca ctcaatggaa 2700
caactcagac gtttctcgaa gacggagacc aagtcacctt ctcaggtgta tgcaaggtat 2760
cagctgatta acacggtttc tgctttagtt taatttgctt tataccccaa caactccaaa 2820
tgaatttcgt tgcatgacat ttcggttaac gcttattaat caaattacgt ctatgattaa 2880
accgttgtag ggagatggtt acaatgttgg gtttggaaca tgcacaggga aaattgttcc 2940
CA 02422760 2003-03-17
'SunGene GmbH & Co. Kga.A 20000445 O.Z. 0817/00017 DE
17
ttcaccgcct tga 2953
<210> 10
<211> 1534
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> CDS
<222> (28)..(1227)
<223> cDNA coding for hydroxyphenylpyruvate dioxygenase
<400> 10
cgagttttag cagagttggt gaaatca atg ggc cac caa aac gcc gcc gtt tca 54
Met Gly His Gln Asn Ala Ala Val Ser
1 5
gag aat caa aac cat gat gac ggc get gcg tcg tcg ccg gga ttc aag 102
Glu Asn Gln Asn His Asp Asp Gly Ala Ala Ser Ser Pro Gly Phe Lys
15 20 25
ctc gtc gga ttt tcc aag ttc gta aga aag aat cca aag tct gat aaa 150
Leu Val Gly Phe Ser Lys Phe Val Arg Lys Asn Pro Lys Ser Asp Lys
30 35 40
ttc aag gtt aag cgc ttc cat cac atc gag ttc tgg tgc ggc gac gca 198
Phe Lys Val Lys Arg Phe His His Ile Glu Phe Trp Cys Gly Asp Ala
45 50 55
acc aac gtc get cgt cgc ttc tcc tgg ggt ctg ggg atg aga ttc tcc 246
Thr Asn Val Ala Arg Arg Phe Ser Trp Gly Leu Gly Met Arg Phe Ser
60 65 70
gcc aaa tcc gat ctt tcc acc gga aac atg gtt cac gcc tct tac cta 294
Ala Lys Ser Asp Leu Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu
75 80 85
ctc acc tcc ggt gac ctc cga ttc Ctt ttc act get cct tac tct ccg 342
Leu Thr Ser Gly Asp Leu Arg Phe Leu Phe Thr Ala Pro Tyr Ser Pro
90 95 100 10S
tct ctc tcc gcc gga gag att aaa ccg aca acc aca get tct atc cca 390
Ser Leu Ser Ala Gly Glu Ile Lys Pro Thr Thr Thr Ala Ser Ile Pro
110 115 120
agt ttc gat cac ggc tct tgt cgt tcc ttc ttc tct tca cat ggt ctc 438
Ser Phe Asp His Gly Ser Cys Arg Ser Phe Phe Ser Sex His Gly Leu
125 130 135
CA 02422760 2003-03-17
~SunGene GmbH & Co. KgaA 20000445 O.Z. 0817/00017 DE
18
ggt gtt aga gcc gtt gcg att gaa gta gaa gac gca gag tca get ttc 486
Gly Val Arg Ala Val Ala Ile Glu Val Glu Asp Ala Glu Ser Ala Phe
140 145 150
tcc atc agt gta get aat ggc get att cct tcg tcg cct cct atc gtc 534
Ser Ile Ser Val Ala Asn Gly Ala Ile Pro Ser Ser Pro Pro Ile Val
155 160 165
ctc aat gaa gca gtt acg atc get gag gtt aaa cta tac ggc gat gtt 582
Leu Asn Glu Ala Val Thr Ile Ala Glu Val Lys Leu Tyr Gly Asp Val
170 175 180 185
gtt ctc cga tat gtt agt tac aaa gca gaa gat acc gaa aaa tcc gaa 630
Val Leu Arg Tyr Val Ser Tyr Lys Ala Glu Asp Thr Glu Lys Ser Glu
190 195 200
ttc ttg cca ggg ttc gag cgt gta gag gat gcg tcg tcg ttc cca ttg 678
Phe Leu Pro Gly Phe Glu Arg Val Glu Asp Ala Ser Ser Phe Pro Leu
205 210 215
gat tat ggt atc cgg cgg ctt gac cac gcc gtg gga aac gtt cct gag 726
Asp Tyr Gly Ile Arg Arg Leu Asp His Ala Val Gly Asn Val Pro Glu
220 225 230
ctt ggt ccg get tta act tat gta gcg ggg ttc act ggt ttt cac caa 774
Leu Gly Pro Ala Leu Thr Tyr Val Ala Gly Phe Thr Gly Phe His Gln
235 240 245
ttc gca gag ttc aca gca gac gac gtt gga acc gcc gag agc ggt tta 822
Phe Ala Glu Phe Thr Ala Asp Asp Val Gly Thr Ala Glu Ser Gly Leu
250 255 260 265
aat tca gcg gtc ctg get agc aat gat gaa atg gtt ctt cta ccg att 870
Asn Ser Ala Val Leu Ala Ser Asn Asp Glu Met Val Leu Leu Pro Ile
270 275 280
aac gag cca gtg cac gga aca aag agg aag agt cag att cag acg tat 918
Asn Glu Pro VaI His Gly Thr Lys Arg Lys Ser Gln Ile Gln Thr Tyr
285 290 295
ttg gaa cat aac gaa ggc gca ggg cta caa cat ctg get ctg atg agt 966
Leu Glu His Asn Glu Gly Ala Gly Leu Gln His Leu Ala Leu Met Ser
300 305 310
gaa gac ata ttc agg acc ctg aga gag atg agg aag agg agc agt att 1014
Glu Asp Ile Phe Arg Thr Leu Arg Glu Met Arg Lys Arg Ser Ser Ile
315 320 325
gga gga ttc gac ttc atg cct tct cct ccg cct act tac tac cag aat 1062
Gly Gly Phe Asp Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Gln Asn
330 335 340 345
CA 02422760 2003-03-17
SunGene GmbH ~ Co. Kga.A 20000445 O.Z. 0817/00017 DE
19
ctc aag aaa cgg gtc ggc gac gtg ctc agc gat gat cag atc aag gag 1110
Leu Lys Lys Arg Val Gly Asp Val Leu Ser Asp Asp Gln Ile Lys Glu
350 355 360
tgt gag gaa tta ggg att ctt gta gac aga gat gat caa ggg acg ttg 1158
Cys Glu Glu Leu Gly Ile Leu Val Asp Arg Asp Asp Gln Gly Thr Leu
365 370 375
ctt caa atc ttc aca aaa cca cta ggt gac agg tac agt tca ttt aat 1206
Leu Gln Ile Phe Thr Lys Pro Leu Gly Asp Arg Tyr Ser Ser Phe Asn
380 385 390
caa aca cat gtt aca gtt ccc taacaatcca tttgatgata aacatgttac 1257
Gln Thr His Val Thr Val Pro
395 400
agtttactaa gcaatctctt gtttatgatt gtgttaatag gccgacgata tttatagaga 1317
taatccagag agtaggatgc atgatgaaag atgaggaagg gaaggcttac cagagtggag 1377
gatgtggtgg tctctgagct cttcaagtcc attgaagaat acgaaaagac tcttgaagcc 1437
aaacagttag tgggatgaac aagaagaaga accaactaaa ggattgtgta attaatgtaa 1497
aactgtttta tcttatcaaa acaatgttat acaacat 1534
<210> 11
<211> 400
<212> PRT
<213> Arabidopsis thaliana
<400> 11
Met Gly His Gln Asn Ala Ala Val Ser Glu Asn Gln Asn His Asp Asp
1 5 10 15
Gly Ala Ala Ser Ser Pro Gly Phe Lys Leu Val Gly Phe Ser Lys Phe
20 25 30
Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys Val Lys Arg Phe His
35 40 45
His Ile Glu Phe Trp Cys Gly Asp Ala Thr Asn Val Ala Arg Arg Phe
50 55 60
Ser Trp Gly Leu Gly Met Arg Phe Ser AIa Lys Ser Asp Leu Ser Thr
65 70 75 80
Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr Ser Gly Asp Leu Arg
85 90 95
Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu Ser Ala Gly Glu Ile
100 105 110
CA 02422760 2003-03-17
SunGene GmbH & Co. KgaA 20000445 O.Z. 0817/00017 DE
Lys Pro Thr Thr Thr Ala Ser Ile Pro Ser Phe Asp His Gly Ser Cys
115 120 125
Arg Ser Phe Phe Ser Ser His Gly Leu Gly Val Arg Ala Val Ala Ile
130 135 140
Glu Val Glu Asp Ala Glu Ser Ala Phe Ser Ile Ser Val Ala Asn Gly
145 150 155 160
Ala Ile Pro Ser Ser Pro Pro Ile Val Leu Asn Glu Ala Val Thr Ile
165 170 175
Ala Glu Val Lys Leu Tyr Gly Asp Val Val Leu Arg Tyr Val Ser Tyr
180 185 190
Lys Ala Glu Asp Thr Glu Lys Ser Glu Phe Leu Pro Gly Phe Glu Arg
195 200 205
Val Glu Asp Ala Ser Ser Phe Pro Leu Asp Tyr Gly Ile Arg Arg Leu
210 215 220
Asp His Ala Val Gly Asn Val Pro Glu Leu Gly Pro Ala Leu Thr Tyr
225 230 235 240
Val Ala Gly Phe Thr Gly Phe His Gln Phe Ala Glu Phe Thr Ala Asp
245 250 255
Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser Ala Val Leu Ala Ser
260 265 270
Asn Asp Glu Met Val Leu Leu Pro Ile Asn Glu Pro Val His Gly Thr
275 280 285
Lys Arg Lys Ser Gln Ile Gln Thr Tyr Leu Glu His Asn Glu Gly Ala
290 295 300
Gly Leu Gln His Leu Ala Leu Met Ser Glu Asp Ile Phe Arg Thr Leu
305 310 315 320
Arg Glu Met Arg Lys Arg Ser Ser Ile Gly Gly Phe Asp Phe Met Pro
325 330 335
Ser Pro Pro Pro Thr Tyr Tyr Gln Asn Leu Lys Lys Arg Val Gly Asp
340 345 350
Val Leu Ser Asp Asp Gln Ile Lys Glu Cys Glu Glu Leu Gly Ile Leu
355 360 365
Val Asp Arg Asp Asp Gln Gly Thr Leu Leu Gln Ile Phe Thr Lys Pro
370 375 380
CA 02422760 2003-03-17
~SunGene GmbH & Co. KgaA 20000445 O.Z. 0817/00017 DE
21
Leu Gly Asp Arg Tyr Ser Ser Phe Asn Gln Thr His Val Thr Val Pro
385 390 395 400
<210> 12
<211> 575
<212> DNA
<213> Brassica napus
<220>
<221> misc feature
<222> (1)..(6)
<223> restriction site
<220>
<221> misc feature
<222> (570)..(575}
<223> restriction site
<220>
<221> misc feature
<222> (7)..(569)
<223> fragment of cDNA coding for
homogentisate-1,2-dioxygenase
<400> 12
gtcgacgggc cgatgggggc gaagggtctt gctgcaccaa gagattttct tgcaccaacg 60
gcatggtttg aggaagggct acggcctgac tacactattg ttcagaagtt tggcggtgaa 120
ctctttactg ctaaacaaga tttctctccg ttcaatgtgg ttgcctggca tggcaattac 180
gtgccttata agtatgacct gcacaagttc tgtccataca acactgtcct tgtagaccat 240
ggagatccat ctgtaaatac agttctgaca gcaccaacgg ataaacctgg tgtggccttg 300
cttgattttg tcatattccc tcctcgttgg ttggttgctg agcatacctt tcgacctcct 360
tactaccatc gtaactgcat gagtgaattt atgggcctaa tctatggtgc ttacgaggcc 420
aaagctgatg gatttctacc tggtggcgca agtcttcaca gttgtatgac acctcatggt 480
ccagatacaa ccacatacga ggcgacgatt gctcgtgtaa atgcaatggc tccttataag 540
ctcacaggca ccatggcctt catgtttgag gtacc 575
<210> 13
<211> 932
<212> DNA
<213> Synechocystis PCC6803
<220>
<221> CDS
<222> (4)..(927)
<223> cDNA coding for homogentisate phytyltransferase
<400> 13
gcc atg gca act atc caa get ttt tgg cgc ttc tcc cgc ccc cat acc 48
Met Ala Thr Ile Gln Ala Phe Trp Arg Phe Ser Arg Pro His Thr
1 5 10 15
CA 02422760 2003-03-17
~SunGene GmbH & Co. KgaA 20000445 O.Z. 0817/00017 DE
22
atc att ggt aca act ctg agc gtc tgg get gtg tat ctg tta act att 96
Ile Ile Gly Thr Thr Leu Ser Val Trp Ala Val Tyr Leu Leu Thr Ile
20 25 30
ctc ggg gat gga aac tca gtt aac tcc cct get tcc ctg gat tta gtg 144
Leu Gly Asp Gly Asn Ser Val Asn Ser Pro Ala Ser Leu Asp Leu Val
35 40 45
ttc ggc get tgg ctg gcc tgc ctg ttg ggt aat gtg tac att gtc ggc 192
Phe Gly Ala Trp Leu Ala Cys Leu Leu Gly Asn Val Tyr Ile Val Gly
50 55 60
ctc aac caa ttg tgg gat gtg gac att gac cgc atc aat aag ccg aat 240
Leu Asn Gln Leu Trp Asp Val Asp Ile Asp Arg Ile Asn Lys Pro Asn
65 70 75
ttg ccc cta get aac gga gat ttt tct atc gcc cag ggc cgt tgg att 288
Leu Pro Leu Ala Asn Gly Asp Phe Ser Ile Ala Gln Gly Arg Trp Ile
80 85 90 95
gtg gga ctt tgt ggc gtt get tcc ttg gcg atc gcc tgg gga tta ggg 336
Val Gly Leu Cys Gly Val Ala Ser Leu Ala Ile Ala Trp Gly Leu Gly
100 105 110
cta tgg ctg ggg cta acg gtg ggc att agt ttg att att ggc acg gcc 384
Leu Trp Leu Gly Leu Thr Val Gly Ile Ser Leu Ile Ile Gly Thr Ala
115 120 125
tat tcg gtg ccg cca gtg agg tta aag cgc ttt tcc ctg ctg gcg gcc 432
Tyr Ser Val Pro Pro Val Arg Leu Lys Arg Phe Ser Leu Leu Ala Ala
130 135 140
ctg tgt att ctg acg gtg cgg gga att gtg gtt aac ttg ggc tta ttt 480
Leu Cys Ile Leu Thr Val Arg Gly Ile Val Val Asn Leu Gly Leu Phe
145 150 155
tta ttt ttt aga att ggt tta ggt tat ccc ccc act tta ata acc ccc 528
Leu Phe Phe Arg Ile Gly Leu Gly Tyr Pro Pro Thr Leu Ile Thr Pro
160 165 170 175
atc tgg gtt ttg act tta ttt atc tta gtt ttc acc gtg gcg atc gcc 576
Ile Trp Val Leu Thr Leu Phe Ile Leu Val Phe Thr Val Ala Ile Ala
180 185 190
att ttt aaa gat gtg cca gat atg gaa ggc gat cgg caa ttt aag att 624
Ile Phe Lys Asp Val Pro Asp Met Glu Gly Asp Arg Gln Phe Lys Ile
195 200 205
caa act tta act ttg caa atc ggc aaa caa aac gtt ttt cgg gga acc 672
Gln Thr Leu Thr Leu Gln Ile Gly Lys Gln Asn Val Phe Arg Gly Thr
210 215 220
CA 02422760 2003-03-17
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tta att tta ctc act ggt tgt tat tta gcc atg gca atc tgg ggc tta 720
Leu Ile Leu Leu Thr Gly Cys Tyr Leu Ala Met Ala Ile Trp Gly Leu
225 230 235
tgg gcg get atg cct tta aat act get ttc ttg att gtt tcc cat ttg 768
Trp Ala Ala Met Pro Leu Asn Thr Ala Phe Leu Ile Val Ser His Leu
240 245 250 255
tgc tta tta gcc tta ctc tgg tgg cgg agt cga gat gta cac tta gaa 816
Cys Leu Leu Ala Leu Leu Trp Trp Arg Ser Arg Asp Val His Leu Glu
260 265 270
agc aaa acc gaa att get agt ttt tat cag ttt att tgg aag cta ttt 864
Ser Lys Thr Glu Ile Ala Ser Phe Tyr Gln Phe Ile Trp Lys Leu Phe
275 280 285
ttc tta gag tac ttg ctg tat ccc ttg get ctg tgg tta cct aat ttt 912
Phe Leu~Glu Tyr Leu Leu Tyr Pro Leu Ala Leu Trp Leu Pro Asn Phe
290 295 300
tct aat act att ttt taggg 932
Ser Asn Thr Ile Phe
305
<210> 14
<211> 308
<212> PRT
<213> Synechocystis PCC6803
<400> 14
Met Ala Thr Ile Gln Ala Phe Trp Arg Phe Ser Arg Pro His Thr Ile
1 5 10 15
Ile Gly Thr Thr Leu Ser Val Trp Ala Val Tyr Leu Leu Thr Ile Leu
20 25 30
Gly Asp Gly Asn Ser Val Asn Ser Pro Ala Ser Leu Asp Leu Val Phe
35 40 45
Gly Ala Trp Leu Ala Cys Leu Leu Gly Asn Val Tyr Ile Val Gly Leu
50 55 60
Asn Gln Leu Trp Asp Val Asp Ile Asp Arg Ile Asn Lys Pro Asn Leu
65 70 75 80
Pro Leu Ala Asn Gly Asp Phe Ser Ile Ala Gln Gly Arg Trp Ile Val
85 90 95
Gly Leu Cys Gly Val Ala Ser Leu Ala Ile Ala Trp Gly Leu Gly Leu
100 105 110
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Trp Leu Gly Leu Thr Val Gly Ile Ser Leu Ile Ile Gly Thr Ala Tyr
115 120 125
Ser Val Pro Pro Val Arg Leu Lys Arg Phe Ser Leu Leu Ala Ala Leu
130 135 140
Cys Ile Leu Thr Val Arg Gly Ile Val Val Asn Leu Gly Leu Phe Leu
145 150 155 160
Phe Phe Arg Ile Gly Leu Gly Tyr Pro Pro Thr Leu Ile Thr Pro Ile
165 170 175
Trp Val Leu Thr Leu Phe Ile Leu Val Phe Thr Val AIa Ile Ala Ile
180 285 190
Phe Lys Asp Val Pro Asp Met Glu Gly Asp Arg Gln Phe Lys Ile Gln
195 200 205
Thr Leu Thr Leu Gln Ile Gly Lys Gln Asn Val Phe Arg Gly Thr Leu
210 215 220
Ile Leu Leu Thr Gly Cys Tyr Leu Ala Met Ala IIe Trp Gly Leu Trp
225 230 235 240
Ala Ala Met Pro Leu Asn Thr Ala Phe Leu Ile Val Ser His Leu Cys
245 250 255
Leu Leu Ala Leu Leu Trp Trp Arg Ser Arg Asp Val His Leu Glu Ser
260 265 270
Lys Thr Glu Ile Ala Ser Phe Tyr Gln Phe Ile Trp Lys Leu Phe Phe
275 280 285
Leu Glu Tyr Leu Leu Tyr Pro Leu AIa Leu Trp Leu Pro Asn Phe Ser
290 295 300
Asn Thr Ile Phe
305
<210> 15
<211> 1159
<212> DNA
<213> Artificial sequence
<220>
<221> CDS
<222> (8)..(1150)
<220>
<221> misc_feature
CA 02422760 2003-03-17
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<222> (1)..(6)
<223> restriction site
<220>
<221> misc_feature
<222> (1154)..(1159)
<223> restriction site
<220>
<223> Description of the artificial sequence: codon usage
optimized cDNA coding for hydroxyphenylpyruvate
dioxygenase from Streptomyces avermitilis
<400> 15
gtcgact atg act caa act act cat cat act cca gat act get aga caa 49
Met Thr Gln Thr Thr His His Thr Pro Asp Thr Ala Arg Gln
1 5 10
get gat cct ttt cca gtt aag gga atg gat get gtt gtt ttc get gtt 97
Ala Asp Pro Phe Pro Val Lys Gly Met Asp Ala Val Val Phe Ala Val
15 20 25 30
gga aac get aag caa get get cat tac tac tct act get ttc gga atg 145
Gly Asn Ala Lys Gln Ala Ala His Tyr Tyr Ser Thr Ala Phe Gly Met
40 45
caa ctt gtt get tac tct gga cca gaa aac gga tct aga gaa act get 193
Gln Leu Val Ala Tyr Ser Gly Pro Glu Asn Gly Ser Arg Glu Thr Ala
50 55 60
tct tac gtt ctt act aac gga tct get aga ttc gtt ctt act tct gtt 241
Ser Tyr Val Leu Thr Asn Gly Ser Ala Arg Phe Val Leu Thr Ser Val
65 70 75
att aag cca get acc cca tgg gga cat ttc ctt get gat cac gtt get 289
Ile Lys Pro Ala Thr Pro Trp Gly His Phe Leu Ala Asp His Val Ala
80 85 90
gaa cac gga gat gga gtt gtt gat ctt get att gaa gtt cca gat get 337
Glu His Gly Asp Gly Val Val Asp Leu Ala Ile Glu Val Pro Asp Ala
95 100 105 110
aga get get cat get tac get att gaa cat gga get aga tct gtt get 385
Arg Ala Ala His Ala Tyr Ala Ile Glu His Gly Ala Arg Ser Val Ala
115 120 125
gaa cca tac gaa ctt aag gat gaa cat gga act gtt gtt ctt get get 433
Glu Pro Tyr Glu Leu Lys Asp Glu His Gly Thr Val Val Leu Ala Ala
130 135 140
CA 02422760 2003-03-17
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att get act tac gga aag act aga cat act ctt gtt gat aga act gga 481
Ile Ala Thr Tyr Gly Lys Thr Arg His Thr Leu Val Asp Arg Thr Gly
145 150 155
tac gat gga cca tac ctt cca gga tac gtt get get get cca att gtt 529
Tyr Asp Gly Pro Tyr Leu Pro Gly Tyr Val Ala Ala Ala Pro Ile Val
160 165 170
gaa cca cca get cat aga acc ttc caa get att gac cat tgt gtt ggt 577
Glu Pro Pro Ala His Arg Thr Phe Gln Ala Ile Asp His Cys Val Gly
175 180 I85 190
aac gtt gaa ctc gga aga atg aac gaa tgg gtt gga ttc tac aac aag 625
Asn Val Glu Leu Gly Arg Met Asn Glu Trp Val Gly Phe Tyr Asn Lys
195 200 205
gtt atg gga ttc act aac atg aag gaa ttc gtt gga gat gat att get 673
Val Met Gly Phe Thr Asn Met Lys Glu Phe Val Gly Asp Asp Ile Ala
210 215 220
act gag tac tct get ctt atg tct aag gtt gtt get gat gga act ctt 721
Thr Glu Tyr Ser Ala Leu Met Ser Lys Val Val Ala Asp Gly Thr Leu
225 230 235
aag gtt aaa ttc cca att aat gaa cca get ctt get aag aag aag tct 769
Lys Val Lys Phe Pro Ile Asn Glu Pro Ala Leu Ala Lys Lys Lys Ser
240 245 250
cag att gat gaa tac ctt gag ttc tac gga gga get gga gtt caa cat 817
Gln Ile Asp Glu Tyr Leu Glu Phe Tyr Gly Gly Ala Gly Val Gln His
255 260 265 270
att get ctt aac act gga gat atc gtg gaa act gtt aga act atg aga 865
Ile Ala Leu Asn Thr Gly Asp Ile Val Glu Thr Val Arg Thr Met Arg
275 280 285
get gca gga gtt caa ttc ctt gat act cca gat tct tac tac gat act 913
Ala Ala Gly Val Gln Phe Leu Asp Thr Pro Asp Ser Tyr Tyr Asp Thr
290 295 300
ctt ggt gaa tgg gtt gga gat act aga gtt cca gtt gat act ctt aga 961
Leu Gly Glu Trp Val Gly Asp Thr Arg Val Pro Val Asp Thr Leu Arg
305 310 315
gaa ctt aag att ctt get gat aga gat gaa gat gga tac ctt ctt caa 1009
Glu Leu Lys Ile Leu Ala Asp Arg Asp Glu Asp Gly Tyr Leu Leu Gln
320 325 330
atc ttc act aag cca gtt caa gat aga cca act gtg ttc ttc gaa atc 1057
Ile Phe Thr Lys Pro Val Gln Asp Arg Pro Thr Val Phe Phe Glu Ile
335 340 345 350
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att gaa aga cat gga tct atg gga ttc gga aag ggt aac ttc aag get 1105
Ile Glu Arg His Gly Ser Met Gly Phe Gly Lys Gly Asn Phe Lys Ala
355 360 365
ctt ttc gaa get att gaa aga gaa caa gag aag aga gga aac ctt 1150
Leu Phe Glu Ala Ile Glu Arg Glu Gln Glu Lys Arg Gly Asn Leu
370 375 380
taggtcgac 1159
<210> 16
<211> 381
<212> PRT
<213> Artificial sequence
<223> Description of the artificial sequence: codon usage
optimized cDNA coding for hydroxyphenylpyruvate
dioxygenase from Streptomyces avermitilis
<400> 16
Met Thr Gln Thr Thr His His Thr Pro Asp Thr Ala Arg Gln Ala Asp
1 5 10 15
Pro Phe Pro Val Lys Gly Met Asp Ala Val Val Phe Ala Val Gly Asn
20 25 30
Ala Lys Gln Ala Ala His Tyr Tyr Ser Thr Ala Phe Gly Met Gln Leu
35 40 45
Val Ala Tyr Ser Gly Pro Glu Asn Gly Ser Arg Glu Thr Ala Ser Tyr
50 55 60
Val Leu Thr Asn Gly Ser Ala Arg Phe Val Leu Thr Ser Val Ile Lys
65 70 75 80
Pro Ala Thr Pro Trp Gly His Phe Leu Ala Asp His Val Ala Glu His
85 90 95
Gly Asp Gly Val Val Asp Leu Ala Ile Glu Val Pro Asp Ala Arg Ala
100 105 110
Ala His Ala Tyr Ala Ile Glu His Gly Ala Arg Ser Val Ala Glu Pro
115 120 125
Tyr Glu Leu Lys Asp Glu His Gly Thr Val Val Leu Ala Ala Ile Ala
130 135 140
Thr Tyr Gly Lys Thr Arg His Thr Leu Val Asp Arg Thr Gly Tyr Asp
145 150 155 160
Gly Pro Tyr Leu Pro Gly Tyr Val Ala Ala Ala Pro Ile Val Glu Pro
165 170 175
CA 02422760 2003-03-17
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Pro Ala His Arg Thr Phe Gln Ala Ile Asp His Cys Val Gly Asn Val
180 185 190
Glu Leu Gly Arg Met Asn Glu Trp Val Gly Phe Tyr Asn Lys Val Met
195 200 205
Gly Phe Thr Asn Met Lys Glu Phe Val Gly Asp Asp Ile Ala Thr Glu
210 215 220
Tyr Ser Ala Leu Met Ser Lys Val Val Ala Asp Gly Thr Leu Lys Val
225 230 235 240
Lys Phe Pro Ile Asn Glu Pro Ala Leu Ala Lys Lys Lys Ser Gln Ile
245 250 255
Asp Glu Tyr Leu Glu Phe Tyr Gly Gly Ala Gly Val Gln His Ile Ala
260 265 270
Leu Asn Thr Gly Asp Ile Val Glu Thr Val Arg Thr Met Arg Ala Ala
275 280 285
Gly Val Gln Phe Leu Asp Thr Pro Asp Ser Tyr Tyr Asp Thr Leu Gly
290 295 300
Glu Trp Val Gly Asp Thr Arg Val Pro Val Asp Thr Leu Arg Glu Leu
305 310 315 320
Lys Ile Leu Ala Asp Arg Asp Glu Asp Gly Tyr Leu Leu Gln Ile Phe
325 330 335
Thr Lys Pro Val Gln Asp Arg Pro Thr Val Phe Phe Glu Ile Ile Glu
340 345 350
Arg His Gly Ser Met Gly Phe Gly Lys Gly Asn Phe Lys Ala Leu Phe
355 360 365
Glu Ala Ile Glu Arg Glu Gln Glu Lys Arg Gly Asn Leu
370 375 380
<210> 17
<211> 815
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> CDS
<222> (37)..(705)
<223> cDNA coding for maleylcetoacetate isomerase
<400> 17
gtaatctccg aagaagaaca aattccttgc tgaatc atg tct tat gtt acc gat 54
CA 02422760 2003-03-17
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Met Ser Tyr Val Thr Asp
1 5
ttt tat cag gcg aag ttg aag ctc tac tct tac tgg aga agc tca tgt 102
Phe Tyr Gln Ala Lys Leu Lys Leu Tyr Ser Tyr Trp Arg Ser Ser Cys
15 20
get cat cgc gtc cgt atc gcc ctc act tta aaa ggg ctt gat tat gaa 150
Ala His Arg Val Arg Ile Ala Leu Thr Leu Lys Gly Leu Asp Tyr Glu
25 30 35
tat ata ccg gtt aat ttg ctc aaa ggg gat caa tcc gat tca gat ttc 198
Tyr Ile Pro Val Asn Leu Leu Lys Gly Asp Gln Ser Asp Ser Asp Phe
40 45 50
aag aag atc aat cca atg ggc act gta cca gcg ctt gtt gat ggt gat 246
Lys Lys Ile Asn Pro Met Gly Thr Val Pro Ala Leu Val Asp Gly Asp
55 60 65 70
gtt gtg att aat gac tct ttc gca ata ata atg tac ctg gat gat aag 294
Val Val Ile Asn Asp Ser Phe Ala Ile Ile Met Tyr Leu Asp Asp Lys
75 80 85
tat ccg gag cca ccg ctg tta cca agt gac tac cat aaa cgg gcg gta 342
Tyr Pro Glu Pro Pro Leu Leu Pro Ser Asp Tyr His Lys Arg Ala Val
90 95 100
aat tac cag gcg acg agt att gtc atg tct ggt ata cag cct cat caa 390
Asn Tyr Gln Ala Thr Ser Ile Val Met Ser Gly Ile Gln Pro His Gln
105 110 115
aat atg get ctt ttt agg tat ctc gag gac aag ata aat get gag gag 438
Asn Met Ala Leu Phe Arg Tyr Leu Glu Asp Lys Ile Asn Ala Glu Glu
120 125 130
aaa act get tgg att act aat get atc aca aaa gga ttc aca get ctc 486
Lys Thr Ala Trp Ile Thr Asn Ala Ile Thr Lys Gly Phe Thr Ala Leu
135 140 145 150
gag aaa ctg ttg gtg agt tgc get gga aaa tac gcg act ggt gat gaa 534
Glu Lys Leu Leu Val Ser Cys Ala Gly Lys Tyr Ala Thr Gly Asp Glu
155 160 165
gtt tac ttg get gat ctt ttc cta gca cca cag atc cac gca gca ttc 582
Val Tyr Leu Ala Asp Leu Phe Leu Ala Pro Gln Ile His Ala Ala Phe
170 175 180
aac aga ttc cat att aac atg gaa cca ttc ccg act ctt gca agg ttt 630
Asn Arg Phe His Ile Asn Met Glu Pro Phe Pro Thr Leu Ala Arg Phe
185 190 195
CA 02422760 2003-03-17
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tac gag tca tac aac gaa ctg cct gca ttt caa aat gca gtc ccg gag 678
Tyr Glu Ser Tyr Asn Glu Leu Pro Ala Phe Gln Asn Ala Val Pro Glu
200 205 210
aag caa cca gat act cct tcc acc atc tgattctgtg aaccgtaagc 725
Lys Gln Pro Asp Thr Pro Ser Thr Ile
215 220
ttctctcagt ctcagctcaa taaaatctct taggaaacaa caacaacacc ttgaacttaa 785
atgtatcata tgaaccagtt tacaaataat 815
<210> 18
<211> 223
<212> PRT
<213> Arabidopsis thaliana
<400> 18
Met Ser Tyr Val Thr Asp Phe Tyr Gln Ala Lys Leu Lys Leu Tyr Ser
1 5 10 15
Tyr Trp Arg Ser Ser Cys Ala His Arg Val Arg Ile Ala Leu Thr Leu
20 25 30
Lys Gly Leu Asp Tyr Glu Tyr Ile Pro Val Asn Leu Leu Lys Gly Asp
40 45
Gln Ser Asp Ser Asp Phe Lys Lys Ile Asn Pro Met Gly Thr Val Pro
50 55 60
Ala Leu Val Asp Gly Asp Val Val Ile Asn Asp Ser Phe Ala Ile Ile
65 70 75 80
Met Tyr Leu Asp Asp Lys Tyr Pro Glu Pro Pro Leu Leu Pro Ser Asp
85 90 95
Tyr His Lys Arg Ala Val Asn Tyr Gln Ala Thr Ser Ile Val Met Ser
100 105 110
Gly Ile Gln Pro His Gln Asn Met Ala Leu Phe Arg Tyr Leu Glu Asp
115 120 125
Lys Ile Asn Ala Glu Glu Lys Thr Ala Trp Ile Thr Asn Ala Ile Thr
130 135 140
Lys Gly Phe Thr Ala Leu Glu Lys Leu Leu Val Ser Cys Ala Gly Lys
145 150 155 160
Tyr Ala Thr Gly Asp Glu Val Tyr Leu Ala Asp Leu Phe Leu Ala Pro
165 170 175
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Gln Ile His Ala Ala Phe Asn Arg Phe His Ile Asn Met Glu Pro Phe
180 185 190
Pro Thr Leu Ala Arg Phe Tyr Glu Ser Tyr Asn Glu Leu Pro Ala Phe
195 200 205
Gln Asn Ala Val Pro Glu Lys Gln Pro Asp Thr Pro Ser Thr Ile
210 215 220
<210> 19
<211> 1350
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> CDS
<222> (63)..(1106)
<223> coding for gamma-tocopherol methyltransferase
<400> 19
ccacgcgtcc gcaaataatc cctgacttcg tcacgtttct ttgtatctcc aacgtccaat 60
as atg aaa gca act cta gca gca ccc tct tct ctc aca agc ctc cct 107
Met Lys Ala Thr Leu Ala Ala Pro Ser Ser Leu Thr Ser Leu Pro
1 5 10 15
tat cga acc aac tct tct ttc ggc tca aag tca tcg ctt ctc ttt cgg 155
Tyr Arg Thr Asn Ser Ser Phe Gly Ser Lys Ser Ser Leu Leu Phe Arg
20 25 30
tct cca tcc tcc tcc tcc tca gtc tct atg acg aca acg cgt gga aac 203
Ser Pro Ser Ser Ser Ser Ser Val Ser Met Thr Thr Thr Arg Gly Asn
35 40 45
gtg get gtg gcg get get get aca tcc act gag gcg cta aga aaa gga 251
Val Ala Val Ala Ala Ala Ala Thr Ser Thr Glu Ala Leu Arg Lys Gly
50 55 60
ata gcg gag ttc tac aat gaa act tcg ggt ttg tgg gaa gag att tgg 299
Ile Ala Glu Phe Tyr Asn Glu Thr Ser Gly Leu Trp Glu Glu Ile Trp
65 70 75
gga gat cat atg cat cat ggc ttt tat gac cct gat tct tct gtt caa 347
Gly Asp His Met His His Gly Phe Tyr Asp Pro Asp Ser Ser Val Gln
80 85 90 95
ctt tct gat tct ggt cac aag gaa get cag atc cgt atg att gaa gag 395
Leu Ser Asp Ser Gly His Lys Glu Ala Gln Ile Arg Met Ile Glu Glu
100 105 110
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tct ctc cgt ttc gcc ggt gtt act gat gaa gag gag gag aaa aag ata 443
Ser Leu Arg Phe Ala Gly Val Thr Asp Glu Glu Glu Glu Lys Lys Ile
115 120 125
aag aaa gta gtg gat gtt ggg tgt ggg att gga gga agc tca aga tat 491
Lys Lys Val Val Asp Val Gly Cys Gly Ile Gly Gly Ser Ser Arg Tyr
130 135 140
ctt gcc tct aaa ttt gga get gaa tgc att ggc att act ctc agc cct 539
Leu Ala Ser Lys Phe Gly Ala Glu Cys Ile Gly Ile Thr Leu Ser Pro
145 150 155
gtt cag gcc aag aga gcc aat gat ctc gcg get get caa tca ctc tct 587
Val Gln Ala Lys Arg Ala Asn Asp Leu Ala Ala Ala Gln Ser Leu Ser
160 165 170 175
cat aag get tcc ttc caa gtt gcg gat gcg ttg gat cag cca ttc gaa 635
His Lys Ala Ser Phe Gln Val Ala Asp Ala Leu Asp Gln Pro Phe Glu.
I80 185 190
gat gga aaa ttc gat cta gtg tgg tcg atg gag agt ggt gag cat atg 683
Asp Gly Lys Phe Asp Leu Val Trp Ser Met Glu Ser Gly Glu His Met
195 200 205
cct gac aag gcc aag ttt gta aaa gag ttg gta cgt gtg gcg get cca 731
Pro Asp Lys Ala Lys Phe Val Lys Glu Leu Val Arg Val Ala Ala Pro
Z10 215 220
gga ggt agg ata ata ata gtg aca tgg tgc cat aga aat cta tct gcg 779
Gly Gly Arg Ile Ile Ile VaI Thr Trp Cys His Arg Asn Leu Ser Ala
225 230 235
ggg gag gaa get ttg cag ccg tgg gag caa aac atc ttg gac aaa atc 827
Gly Glu Glu Ala Leu Gln Pro Trp Glu Gln Asn Ile Leu Asp Lys Ile
240 245 250 255
tgt aag acg ttc tat ctc ccg get tgg tgc tcc acc gat gat tat gtc 875
Cys Lys Thr Phe Tyr Leu Pro Ala Trp Cys Ser Thr Asp Asp Tyr Val
260 265 270
aac ttg ctt caa tcc cat tct ctc cag gat att aag tgt gcg gat tgg 923
Asn Leu Leu Gln Ser His Ser Leu Gln Asp Ile Lys Cys Ala Asp Trp
275 280 285
tca gag aac gta get cct ttc tgg cct gcg gtt ata cgg act gca tta 971
Ser Glu Asn Val Ala Pro Fhe Trp Pro Ala Val Ile Arg Thr Ala Leu
290 295 300
aca tgg aag ggc ctt gtg tct ctg ctt cgt agt ggt atg aaa agt att 1019
Thr Trp Lys Gly Leu Val Ser Leu Leu Arg Ser Gly Met Lys Ser Ile
305 310 315
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aaa gga gca ttg aca atg cca ttg atg att gaa ggt tac aag aaa ggt 1067
Lys Gly Ala Leu Thr Met Pro Leu Met Ile Glu Gly Tyr Lys Lys Gly
320 325 330 335
gtc att aag ttt ggt atc atc act tgc cag aag cca ctc taagtctaaa 1116
Val Ile Lys Phe Gly Ile Ile Thr Cys Gln Lys Pro Leu
340 345
gctatactag gagattcaat aagactataa gagtagtgtc tcatgtgaaa gcatgaaatt 1176
ccttaaaaac gtcaatgtta agcctatgct tcgttatttg ttttagataa gtatcatttc 1236
actcttgtct aaggtagttt ctataaacaa taaataccat gaattagctc atgttatctg 1296
gtaaattctc ggaagtgatt gtcatggatt aactcaaaaa aaaaaaaaaa aaaa 1350
<210> 20
<211> 348
<212> PRT
<213> Arabidopsis thaliana
<400> 20
Met Lys Ala Thr Leu Ala Ala Pro Ser Ser Leu Thr Ser Leu Pro Tyr
1 5 10 15
Arg Thr Asn Ser Ser Phe Gly Ser Lys Ser Ser Leu Leu Phe Arg Ser
20 25 30
Pro Ser Ser Ser Ser Ser Val Ser Met Thr Thr Thr Arg Gly Asn Val
35 40 45
Ala Val Ala Ala Ala Ala Thr Ser Thr Glu Ala Leu Arg Lys Gly Ile
50 55 60
Ala Glu Phe Tyr Asn Glu Thr Ser Gly Leu Trp Glu Glu Ile Trp Gly
65 70 75 80
Asp His Met His His Gly Phe Tyr Asp Pro Asp Ser Ser Val Gln Leu
85 90 95
Ser Asp Ser Gly His Lys Glu Ala Gln Ile Arg Met Ile Glu Glu Ser
100 105 110
Leu Arg Phe Ala Gly Val Thr Asp Glu Glu Glu Glu Lys Lys Ile Lys
115 120 125
Lys Val Val Asp Val Gly Cys Gly Ile Gly Gly Ser Ser Arg Tyr Leu
130 135 140
Ala Ser Lys Phe Gly Ala Glu Cys Ile Gly Ile Thr Leu Ser Pro Val
145 150 155 160
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Gln Ala Lys Arg Ala Asn Asp Leu Ala Ala Ala Gln Ser Leu Ser His
165 170 175
Lys Ala Ser Phe Gln Val Ala Asp Ala Leu Asp Gln Pro Phe Glu Asp
180 185 190
Gly Lys Phe Asp Leu Val Trp Ser Met Glu Ser Gly Glu His Met Pro
195 200 205
Asp Lys Ala Lys Phe Val Lys Glu Leu Val Arg Val Ala Ala Pro Gly
210 215 220
Gly Arg Ile Ile Ile Val Thr Trp Cys His Arg Asn Leu Ser Ala Gly
225 230 235 240
Glu Glu Ala Leu Gln Pro Trp Glu Gln Asn Ile Leu Asp Lys Ile Cys
245 250 255
Lys Thr Phe Tyr Leu Pro Ala Trp Cys Ser Thr Asp Asp Tyr Val Asn
260 265 270
Leu Leu Gln Ser His Ser Leu Gln Asp Ile Lys Cys Ala Asp Trp Ser
275 280 285
Glu Asn Val Ala Pro Phe Trp Pro Ala Val Ile Arg Thr Ala Leu Thr
290 295 300
Trp Lys Gly Leu Val Ser Leu Leu Arg Ser Gly Met Lys Ser Ile Lys
305 310 315 320
Gly Ala Leu Thr Met Pro Leu Met Ile Glu Gly Tyr Lys Lys Gly Val
325 330 335
Ile Lys Phe Gly Ile Ile Thr Cys Gln Lys Pro Leu
340 345
<210> 21
<211> 957
<212> DNA
<213> Synechocystis PCC6803
<220>
<221> CDS
<222> (1)..(954)
<223> cDNA coding for 2-methyl-6-phytylhydrochinone
methyltransferase
<400> 21
atg ccc gag tat ttg ctt ctg ccc get ggc cta att tcc ctc tcc ctg 48
Met Pro Glu Tyr Leu Leu Leu Pro Ala Gly Leu Ile Ser Leu Ser Leu
1 5 10 15
CA 02422760 2003-03-17
SunGene GmbH & Co. Kga.A 20000445 O.Z. 0817/00017 DE
gcg atc gcc get gga ctg tat ctc cta act gcc cgg ggc tat cag tca 96
Ala Ile Ala Ala Gly Leu Tyr Leu Leu Thr Ala Arg Gly Tyr Gln Ser
20 25 30
tcg gat tcc gtg gcc aac gcc tac gac caa tgg aca gag gac ggc att 144
Ser Asp Ser Val Ala Asn Ala Tyr Asp Gln Trp Thr Glu Asp Gly Ile
35 40 45
ttg gaa tat tac tgg ggc gac cat atc cac ctc ggc cat tat ggc gat 192
Leu Glu Tyr Tyr Trp Gly Asp His Ile His Leu Gly His Tyr Gly Asp
50 55 60
ccg cca gtg gcc aag gat ttc atc caa tcg aaa att gat ttt gtc cat 240
Pro Pro Val Ala Lys Asp Phe Ile Gln Ser Lys Ile Asp Phe Val His
65 70 75 80
gcc atg gcc cag tgg ggc gga tta gat aca ctt ccc ccc ggc aca acg 288
Ala Met Ala Gln Trp Gly G1y Leu Asp Thr Leu Pro Pro Gly Thr Thr
85 90 95
gta ttg gat gtg ggt tgc ggc att ggc ggt agc agt cgc att ctc gcc 336
Val Leu Asp Val Gly Cys Gly Ile Gly Gly Ser Ser Arg Ile Leu Ala
100 105 110
aaa gat tat ggt ttt aac gtt acc ggc atc acc att agt ccc caa cag 384
Lys Asp Tyr Gly Phe Asn Val Thr Gly Ile Thr Ile Ser Pro Gln Gln
115 120 125
gtg aaa cgg gcg acg gaa tta act cct ccc gat gtg acg gcc aag ttt 432
Val Lys Arg Ala Thr Glu Leu Thr Pro Pro Asp Val Thr Ala Lys Phe
130 135 140
gcg gtg gac gat get atg get ttg tct ttt cct gac ggt agt ttc gac 480
Ala Val Asp Asp Ala Met Ala Leu Ser Phe Pro Asp Gly Ser Phe Asp
145 150 155 160
gta gtt tgg tcg gtg gaa gca ggg ccc cac atg cct gac aaa get gtg 528
Val Val Trp Ser Val Glu Ala Gly Pro His Met Pro Asp Lys Ala Val
165 170 175
ttt gcc aag gaa tta ctg cgg gtc gtg aaa cca ggg ggc att ctg gtg 576
Phe Ala Lys Glu Leu Leu Arg Val Val Lys Pro Gly Gly Ile Leu Val
180 185 190
gtg gcg gat tgg aat caa cgg gac gat cgc caa gtg ccc ctc aac ttc 624
Val Ala Asp Trp Asn Gln Arg Asp Asp Arg Gln Val Pro Leu Asn Phe
195 200 205
tgg gaa aaa cca gtg atg cga caa ctg ttg gat caa tgg tcc cac cct 672
Trp Glu Lys Pro Val Met Arg Gln Leu Leu Asp Gln Trp Ser His Pro
210 215 220
CA 02422760 2003-03-17
SunGene GmbH & Co. Kga.A 20000445 O.Z. 0817/00017 DE
36
gcc ttt gcc agc att gaa ggt ttt gcg gaa aat ttg gaa gcc acg ggt 720
Ala Phe Ala Ser Ile Glu Gly Phe Ala GIu Asn Leu Glu Ala Thr Gly
225 230 235 240
ttg gtg gag ggc cag gtg act act get gat tgg act gta ceg acc ctc 768
Leu Val Glu Gly Gln Val Thr Thr Ala Asp Trp Thr Val Pro Thr Leu
245 250 255
ccc get tgg ttg gat acc att tgg cag ggc att atc cgg ccc cag ggc 816
Pro Ala Trp Leu Asp Thr Ile Trp Gln Gly Ile Ile Arg Pro Gln Gly
260 265 270
tgg tta caa tac ggc att cgt ggg ttt atc aaa tcc gtg cgg gaa gta 864
Trp Leu Gln Tyr Gly Ile Arg Gly Phe Ile.Lys Ser Val Arg Glu Val
275 280 285
ccg act att tta ttg atg cgc ctt gcc ttt ggg gta gga ctt tgt cgc 912
Pro Thr Ile Leu Leu Met Arg Leu Ala Phe Gly Val Gly Leu Cys Arg
290 295 300
ttc ggt atg ttc aaa gca gtg cga aaa aac gcc act caa get taa 957
Phe Gly Met Phe Lys Ala Val Arg Lys Asn Ala Thr Gln Ala
305 310 315
<210> 22
<211> 318
<212> PRT
<213> Synechocystis PCC6803
<400> 22
Met Pro Glu Tyr Leu Leu Leu Pro Ala Gly Leu Ile Ser Leu Ser Leu
1 5 10 15
Ala Ile Ala Ala Gly Leu Tyr Leu Leu Thr Ala Arg Gly Tyr Gln Ser
20 25 30
Ser Asp Ser Val Ala Asn Ala Tyr Asp Gln Trp Thr Glu Asp Gly Ile
35 40 45
Leu Glu Tyr Tyr Trp Gly Asp His Ile His Leu Gly His Tyr Gly Asp
50 55 60
Pro Pro Val Ala Lys Asp Phe Ile Gln Ser Lys Ile Asp Phe Val His
65 70 75 80
Ala Met Ala Gln Trp Gly Gly Leu Asp Thr Leu Pro Pro Gly Thr Thr
85 90 95
Val Leu Asp Val Gly Cys Gly Ile Gly Gly Ser Ser Arg Ile Leu Ala
100 105 110
CA 02422760 2003-03-17
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37
Lys Asp Tyr Gly Phe Asn Val Thr Gly Ile Thr Ile Ser Pro Gln Gln
1I5 120 125
Val Lys Arg Ala Thr Glu Leu Thr Pro Pro Asp Val Thr Ala Lys Phe
130 135 140
Ala Val Asp Asp Ala Met Ala Leu Ser Phe Pro Asp Gly Ser Phe Asp
145 150 155 160
Val Val Trp Ser Val Glu Ala Gly Pro His Met Pro Asp Lys Ala Val
165 170 175
Phe Ala Lys Glu Leu Leu Arg Val Val Lys Pro Gly Gly Ile Leu Val
180 185 190
Val Ala Asp Trp Asn Gln Arg Asp Asp Arg Gln Val Pro Leu Asn Phe
195 200 205
Trp Glu Lys Pro Val Met Arg Gln Leu Leu Asp Gln Trp Ser His Pro
210 215 220
Ala Phe Ala Ser Ile Glu Gly Phe Ala Glu Asn Leu Glu Ala Thr Gly
225 230 235 240
Leu Val Glu Gly Gln Val Thr Thr Ala Asp Trp Thr Val Pro Thr Leu
245 250 255
Pro Ala Trp Leu Asp Thr Ile Trp Gln Gly Ile Ile Arg Pro Gln Gly
260 265 270
Trp Leu Gln Tyr Gly Ile Arg Gly Phe Ile Lys Ser Val Arg Glu Val
275 280 285
Pro Thr Ile Leu Leu Met Arg Leu Ala Phe Gly Val Gly Leu Cys Arg
290 295 300
Phe Gly Met Phe Lys Ala Val Arg Lys Asn Ala Thr Gln Ala
305 310 315
<210> 23
<211> 1395
<212> DNA
<213> Nicotiana tabacum
<220>
<221> CDS
<222> (1)..(1392)
<223> cDNA coding for geranylgeranylpyrophosphate
oxidoreductase
CA 02422760 2003-03-17
SunGeae GmbH & Co. Kga.A 20000445 O.Z. 0827/00017 DE
38
<400> 23
atg get tcc att get ctc aaa act ttc ace ggc cte cgt eaa tcc tcg 48
Met Ala Ser Ile Ala Leu Lys Thr Phe Thr Gly Leu Arg Gln Ser Ser
1 5 10 15
ccg gaa aac aat tcc att act ctt tct aaa tcc ctc ccc ttc acc caa 96
Pro Glu Asn Asn Ser Ile Thr Leu Ser Lys Ser Leu Pro Phe Thr Gln
20 25 30
acc cac cgt agg ctc cga atc aat get tcc aaa tcc agc cca aga gte 144
Thr His Arg Arg Leu Arg Ile Asn Ala Ser Lys Ser Ser Pro Arg Val
35 40 45
aac gge cgc aat ctt cgt gtt gcg gtg gtg ggc ggt ggt ect get ggt 192
Asn Gly Arg Asn Leu Arg Val Ala Val Val Gly Gly Gly Pro Ala Gly
50 55 60
ggc gcc gcc get gaa aca ctc gec aag gga gga att gaa acc ttc tta 240
Gly Ala Ala Ala Glu Thr Leu Ala Lys Gly Gly Ile Glu Thr Phe Leu
65 70 75 80
atc gaa cgc aaa atg gac aac tgc aaa ccc tgc ggt ggg gcc atc cca 288
Ile Glu Arg Lys Met Asp Asn Cys Lys Pro Cys Gly Gly Ala Ile Pro
85 90 95
ctt tgc atg gtg gga gaa ttt gac ctc cct ttg gat atc att gac cgg 336
Leu Cys Met Val Gly Glu Phe Asp Leu Pro Leu Asp Ile Ile Asp Arg
100 105 110
aaa gtt aca aag atg aag atg att tcc cca tec aac gtt get gtt gat 384
Lys Val Thr Lys Met Lys Met Ile Ser Pro Ser Asn Val Ala Val Asp
115 120 125
att ggt cag act tta aag cct cac gag tac atc ggt atg gtg cgc cgc 432
Ile Gly Gln Thr Leu Lys Pro His Glu Tyr Ile Gly Met Val Arg Arg
130 135 140
gaa gta ctc gat get tac cto cgt gac cgc get get gaa gcc gga gcc 480
Glu Val Leu Asp Ala Tyr Leu Arg Asp Arg Ala Ala Glu Ala Gly Ala
145 150 155 160
tct gtt ctc aac ggc ttg ttc ctc aaa atg gac atg ece aaa get ccc 528
Ser Val Leu Asn Gly Leu Phe Leu Lys Met Asp Met Pro Lys Ala Pro
165 170 175
aac gca cct tae gtc ett cae tac aca get tac gac tee aaa act aat 576
Asn Ala Pro Tyr Val Leu His Tyr Thr Ala Tyr Asp Ser Lys Thr Asn
180 185 190
CA 02422760 2003-03-17
SunGene GmbH & Co. KgaA 20000445 O.Z. 0817/0001? DE
39
ggc gcg ggg gag aag cgt acc ctg gaa gtt gac gcc gtt atc ggc get 624
Gly Ala Gly Glu Lys Arg Thr Leu Glu Val Asp Ala Val Ile Gly Ala
295 200 205
gac ggt gca aat tcc cgt gtc gca aaa tcc ata aac gcc ggt gac tac 672
Asp Gly Ala Asn Ser Arg Val Ala Lys Ser Ile Asn Ala Gly Asp Tyr
210 215 220
gag tac get att gca ttc caa gaa agg att aaa att tcc gat gat aaa 720
Glu Tyr Ala Ile Ala Phe Gln Glu Arg Ile Lys Ile Ser Asp Asp Lys
225 230 235 240
atg aag tat tac gag aat tta get gaa atg tac gtg ggt gat gac gtg 768
Met Lys Tyr Tyr Glu Asn Leu Ala Glu Met Tyr Val Gly Asp Asp Val
245 250 255
tcc cct gat ttt tac ggg tgg gtt ttc ccc aaa tgt gac cac gtt gcc 816
Ser Pro Asp Phe Tyr Gly Trp Val Phe Pro Lys Cys Asp His Val Ala
260 265 270
gtt ggc act ggc aca gtc acc cac aaa get gac atc aaa aaa ttc cag 864
Val Gly Thr Gly Thr Val Thr His Lys Ala Asp IIe Lys Lys Phe Gln
27S 280 285
cta get aca aga ttg aga get gat tcc aaa atc acc ggc gga aaa att 912
Leu Ala Thr Arg Leu Arg Ala Asp Ser Lys Ile Thr Gly Gly Lys Ile
290 295 300
atc cgg gtc gag gcc cac ccg att cca gaa cac cca aga ccc aga aga 960
I1e Arg Val Glu Ala His Pro Ile Pro Glu His Pro Arg Pro Arg Arg
305 310 315 320
tta caa gac aga gtt gca ttg gtt ggt gat gcg gca ggg tac gtg acc 1008
Leu Gln Asp Arg Val Ala Leu Val Gly Asp Ala Ala Gly Tyr Val Thr
325 330 335
aaa tgt tcg ggc gaa ggg att tac ttc gcg gca aag agt gga cgt atg 1056
Lys Cys Ser Gly Glu Gly Ile Tyr Phe Ala Ala Lys Ser Gly Arg Met
340 345 350
tgt get gaa gca att gtt gaa ggg tca gaa atg gga aaa aga atg gtg 1104
Cys Ala Glu Ala Ile Val Glu Gly Ser Glu Met Gly Lys Arg Met Val
355 360 365
gac gag agt gat ttg agg aag tat ttg gag aaa tgg gac aag act tat 1152
Asp Glu Ser Asp Leu Arg Lys Tyr Leu Glu Lys Trp Asp Lys Thr Tyr
370 375 380
tgg cca acg tac aag gtg ctt gat ata ttg cag aag gta ttt tac agg 1200
Trp Pro Thr Tyr Lys VaI Leu Asp Ile Leu Gln Lys Val Phe Tyr Arg
385 390 395 400
CA 02422760 2003-03-17
SunGene GmbH & Co. Kga.A 20000445 O.Z. 0817/00017 DE
tcg aat ccg gcg agg gaa gca ttt gtt gaa atg tgc gca gat gag tat 1248
Ser Asn Pro Ala Arg GIu Ala Phe Val Glu Met Cys Ala Asp Glu Tyr
405 410 415
gtg cag aag atg aca ttt gac agc tat ttg tac aag aaa gta gca cca 1296
Val Gln Lys Met Thr Phe Asp Ser Tyr Leu Tyr Lys Lys Val Ala Pro
420 425 430
gga aac cca att gaa gac ttg aag ctt get gtg aat acc att gga agt 1344
Gly Asn Pro Ile Glu Asp Leu Lys Leu Ala Val Asn Thr Ile GIy Ser
435 440 445
ttg gtg aga get aat gca cta aga agg gaa atg gac aag ctc agt gta 1392
Leu Val Arg Ala Asn Ala Leu Arg Arg Glu Met Asp Lys Leu Ser Val
450 455 460
taa 1395
<210>24
<211>464
<212>PRT
<213>Nicotiana tabacum
<400> 24
Met Ala Ser Ile Ala Leu Lys Thr Phe Thr Gly Leu Arg Gln Ser Ser
1 5 10 15
Pro Glu Asn Asn Ser Ile Thr Leu Ser Lys Ser Leu Pro Phe Thr Gln
20 25 30
Thr His Arg Arg Leu Arg Ile Asn Ala Ser Lys Ser Ser Pro Arg Val
35 40 45
Asn Gly Arg Asn Leu Arg Val Ala Val Val Gly Gly Gly Pro Ala Gly
55 60
Gly Ala Ala Ala Glu Thr Leu Ala Lys Gly Gly Ile Glu Thr Phe Leu
65 70 75 80
Ile Glu Arg Lys Met Asp Asn Cys Lys Pro Cys Gly Gly Ala Ile Pro
85 90 95
Leu Cys Met Val Gly Glu Phe Asp Leu Pro Leu Asp Ile Ile Asp Arg
100 105 110
Lys Val Thr Lys Met Lys Met Ile Ser Pro Ser Asn Val Ala Val Asp
115 120 125
Ile Gly Gln Thr Leu Lys Pro His Glu Tyr Ile Gly Met Val Arg Arg
130 135 140
CA 02422760 2003-03-17
SunGene GmbH & Co. KgaA 20000445 O.Z. 0817/00017 DE
41
Glu Val Leu Asp Ala Tyr Leu Arg Asp Arg Ala Ala Glu Ala Gly Ala
145 150 155 160
Ser Val Leu Asn Gly Leu Phe Leu Lys Met Asp Met Pro Lys Ala Pro
165 170 175
Asn Ala Pro Tyr Val Leu His Tyr Thr Ala Tyr Asp Ser Lys Thr Asn
180 185 190
Gly Ala Gly Glu Lys Arg Thr Leu Glu Val Asp Ala Val Ile Gly Ala
195 200 205
Asp Gly Ala Asn Ser Arg Val Ala Lys Ser Ile Asn Ala Gly Asp Tyr
210 215 220
Glu Tyr Ala Ile Ala Phe Gln Glu Arg Ile Lys Ile Ser Asp Asp Lys
225 230 235 240
Met Lys Tyr Tyr Glu Asn Leu Ala Glu Met Tyr Val Gly Asp Asp Val
245 250 255
Ser Pro Asp Phe Tyr Gly Trp Val Phe Pro Lys Cys Asp His Val Ala
260 265 270
Val Gly Thr Gly Thr Val Thr His Lys Ala Asp Ile Lys Lys Phe Gln
275 280 285
Leu Ala Thr Arg Leu Arg Ala Asp Ser Lys Ile Thr Gly Gly Lys Ile
290 295 300
Ile Arg Val Glu Ala His Pro Ile Pro Glu His Pro Arg Pro Arg Arg
305 310 315 320
Leu Gln Asp Arg Val Ala Leu Val Gly Asp Ala Ala Gly Tyr Val Thr
325 330 335
Lys Cys Ser Gly Glu Gly Ile Tyr Phe Ala Ala Lys Ser Gly Arg Met
340 345 350
Cys Ala Glu Ala Ile Val Glu Gly Ser Glu Met Gly Lys Arg Met Val
355 360 365
Asp Glu Ser Asp Leu Arg Lys Tyr Leu Glu Lys Trp Asp Lys Thr Tyr
370 375 380
Trp Pro Thr Tyr Lys Val Leu Asp Ile Leu Gln Lys Val Phe Tyr Arg
385 390 395 400
Ser Asn Pro Ala Arg Glu Ala Phe Val Glu Met Cys Ala Asp Glu Tyr
405 410 415
CA 02422760 2003-03-17
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42
Val Gln Lys Met Thr Phe Asp Ser Tyr Leu Tyr Lys Lys Val Ala Pro
420 425 430
Gly Asn Pro Ile Glu Asp Leu Lys Leu Ala Val Asn Thr Ile Gly Ser
435 440 445
Leu Val Arg Ala Asn Ala Leu Arg Arg Glu Met Asp Lys Leu Ser Val
450 455 460
<210> 25
<211> 26
<212> DNA
<213> oligonucleotide
<400> 25
gtcgacggnc cnatnggngc naangg 26
<210> 26
<211> 24
<212> DNA
<213> oligonucleotide
<400> 26
aagcttccga tctagtaaca taga 24
<210> 27
<211> 32
<212> DNA
<213> oligonucleotide
<400> 27
attctagaca tggagtcaaa gattcaaata ga 32
<210> 28
<211> 32
<212> DNA
<213> oligonucleotide
<400> 28
attctagagg acaatcagta aattgaacgg ag 32
<210> 29
<211> 26
<212> DNA
<213> oligonucleotide
<400> 29
atgtcgacat gtcttatgtt accgat
26
<210> 30
<211> 25
CA 02422760 2003-03-17
SunGene GmbH & Co. Kga.A 20000445 O.Z. 0817/000i7 DE
a
43
<212> DNA
<213> oligonucleotide
<400> 30
atggatccct ggttcatatgataca 25
<210> 31
<211> 26
<212> DNA
<213> o2igonucleotide
<400> 31
atgtcgacgg aaactctgaaccatat 26
<210> 32
<211> 25
<212> DNA
<213> oligonucleotide
<400> 32
atggtaccga atgtgatgcctaagt 25
<210> 33
<211> 29
<212> DNA
<213> oligonucleotide
<400> 33
ggtacctcra acatraangccatngtncc 29
<210> 34
<211> 25
<212> DNA
<213> oligonucleotide
<400> 34
gaattcgatc tgtcgtctcaaactc 25
<210> 35
<211> 26
<212> DNA
<213> oligonucleotide
<400> 35
ggtaccgtga tagtaaacaactaatg 26
<210> 36
<211> 34
<212> DNA
<213> oligonucleotide
CA 02422760 2003-03-17
Asn Gly Arg Asn Leu Arg Val Ala Val Val Gly Gly Gly Pro Ala Gly
55 60
Gly Ala Ala Ala Glu Thr Leu Ala Lys G
SunGene GmbH & Co. KgaA 20000445 O.Z. 0817/00017 DE
44
<400> 36
atggtacctt ttttgcataa acttatcttc atag 34
<210> 37
<211> 43
<212> DNA
<213> oligonucleotide
<400> 37
atgtcgaccc gggatccagg gccctgatgg gtcccatttt ccc
43
<210> 38
<211> 25
<212> DNA
<213> oligonucleotide
<400> 38
gtcgacgaat ttccccgaat cgttc 25
<210> 39
<211> 24
<212> DNA
<213> oligonucleotide
<400> 39
aagcttccga tctagtaaca taga 24
<210> 40
<211> 25
<212> DNA
<213> oligonucleotide
<400> 40
aagcttgatc tgtcgtctca aactc 25
<210> 41
<211> 1721
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> misc_feature
<222> (1)..(8)
<223> restriction site linker
<220>
<22I> misc_feature
<222> (1714)..(1721)
<223> restriction site linker
CA 02422760 2003-03-17
SunGene GmbH & Co. KgaA 20000445 O.Z. 0817/00017 DE
<220>
<221> misc feature
<222> (9)..(1713)
<223> fragment from gene coding for maleylacetoactetate
isomerase (MA.AI)
<400> 41
atgtcgacat gtcttatgtt accgattttt atcaggcgaa gttgaagctc tactcttact 60
ggagaagctc atgtgctcat cgcgtccgta tcgccctcac tttaaaaggt accagccaat 120
gattttattc ttttcttgtg agcaattctt tgatctgaat ttggttcttg ttcgattttc 180
attagggctt gattatgaat atataccggt taatttgctc aaaggggatc aatccgattc 240
aggtgcgtag tttctaggtt atattgaact ttatttgaag taacattgta aagataagaa 300
tggtaagtaa ctgagatttc ttatgttaga cttagaagtt tattcgtttt ggttctctag 360
atttcaagaa gatcaatcca atgggcactg taccagcgct tgttgatggt gatgttgtga 420
ttaatgactc tttcgcaata ataatggtca gtagtaacac atccatttag tttgtttggt 480
tttgttgatg aaaaggaaca ttcgtttatt cgtcttgttg tttttcaaat ggacagtacc 540
tggatgataa gtatccggag ccaccgctgt taccaagtga ctaccataaa cgggcggtaa 600
attaccaggt atcttcgatc ctttgtcttc agatgatgat gtgttgccat catctgcaaa 660
accatgtagt taagtccaaa tgtagtgaac attatcagct ttagattgcg agtgtgatcg 720
ttgttcttat tttgtatatt tcaggcgacg agtattgtca tgtctggtat acagcctcat 780
caaaatatgg ctctttttgt gagaagatga gattaatgta atggattcta ctaatggagg 840
ttctataaca aagcaaacat agttacattt tgtcattttt tttaacagag gtatctcgag 900
gacaagataa atgctgagga gaaaactgct tggattacta atgctatcac aaaaggattc 960
acaggtatga tatctctaat ctacctatac gtaatcaaga accaagacat atgttcaaaa 1020
tgtgattttg ttgatattgt ggttgtacag gtttataacg acctgtctga taatgtctca 1080
tatgtccttc agctctcgag aaactgttgg tgagttgcgc tggaaaatac gcgactggtg 1140
atgaagttta cttggtatgt ctctaaatct ccctggataa tctctatggt actactctct 1200
tctttattac aatgaagcat tgttttgcag gctgatcttt tcctagcacc acagatccac 1260
gcagcattca acagattcca tattaacatg gtacttttcc tcagctaatc tcttctcctg 1320
gtacctagat attgcattgt atatcccccc aaattccatg gaatccttga tcagagtttt 1380
aaggtagcat gaaccaaatg ttatctctgt ctcacacttt cacattcaca gagtaacata 1440
gacgtaatac tcagtttcat aacttttttt cctcgcatca cttggttttc atctctacaa 1500
ttttgttgta taggaaccat tcccgactct tgcaaggttt tacgagtcat acaacgaact 1560
gcctgcattt caaaatgcag tcccggagaa gcaaccagat actccttcca ccatctgatt 1620
ctgtgaaccg taagcttctc tcagtctcag ctcaataaaa tctcttagga aacaacaaca 1680
acaccttgaa cttaaatgta tcatatgaac cagggatcca t 1721
<210> 42
<211> 622
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> misc feature
<222> (1)..(8)
<223> restriction site linker
<220>
<221> misc feature
<222> (615)..(622)
<223> restriction site linker
CA 02422760 2003-03-17
SunGene GmbH & Co. KgaA 20000445 O.Z. 0817/00017 DE
E.
J
46
<220>
<221> misc_feature
<222> (9)..(614)
<223> fragment from gene coding for fumarylacetoacetate
hydrolase (FAAH)
<400> 42
atgtcgacgg aaactctgaa ccatattttg gaccttcgaa gaaacttgat tttgagcttg 60
agatggtaag catctgatgc ctcagttatg tggatttgtt ttacaatgat tcggttgatg 120
ctttttggtg ctagttaaga ataacggcat tgacaaacct ctcttttatc acatgatatt 180
caggctgctg tggttggtcc aggaaatgaa ttgggaaagc ctattgacgt gaataatgca 240
gccgatcata tatttggtct attactgatg aatgactgga gtggtactca cttaactata 300
gttttcgttg agtcatcttt aacctgaccg ggcatgaccg gtttttttaa atgtttgttg 360
ttatagctag ggatattcag gcgtgggagt atgtacctct tggtcctttc ctggggaaga 420
gttttggtga gatatttggc ttcaatactt tgatttcatt tcctctagtt gaagtatatg 480
ggcaaagaac ttcggtgaat gttgtcttgt tgtgttgtag ggactactat atccccttgg 540
attgttacct tggatgcgct tgagcctttt ggttgtcaag ctcccaagca ggttggtact 600
taggcatcac attcggtacc at 622
<210> 43
<211> 32
<212> DNA
<213> oligonucleotide
<400> 43
atgaattcca tggagtcaaa gattcaaata ga 32
<210> 44
<211> 32
<212> DNA
<213> oligonucleotide
<400> 44
atgaattcgg acaatcagta aattgaacgg ag 32
CA 02422760 2003-03-17
